Anti-cancer peptides, and methods and uses thereof

Abstract

Provided herein are anticancer peptides (ACPs). methods of generating such ACPs, methods of killing cancer cells using such ACPs, and methods of treating a patient in need thereof using such ACPs.

Description

ANTI-CANCER PEPTIDES, AND METHODS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States provisional patent application No. 63 / 578,506, filed on August 24, 2023, and to United States provisional patent application No. 65 / 587,773, filed on October 4, 2023, each of which are incorporated by reference in their entireties herein.SEQUENCE LISTING

[0002] This application incorporates by reference the material in the Extensible Markup Language (XML) file titled IUIC133_SEQ_LISTING.XML, which was created on August 22, 2024 and is 24.543 bytes in size.FIELD

[0003] Embodiments disclosed herein are directed to anti-cancer peptides, methods of generating thereof, and methods of use thereof.BACKGROUND

[0004] Cancer is characterized by proliferation of abnormal cells. Many cancer treatments include painful surgeries, as well as chemotherapies and radiation therapies with undesirable side effects. An ongoing, urgent need exists for new therapeutic interventions for cancer.SUMMARY

[0005] This summary' is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter. Furthermore, it is envisioned that alternative embodiments may combine features of two or more of the below-summarized embodiments. Further embodiments, forms, features, and aspects of the present application shall become apparent from the description and figures provided herewith.

[0006] In an aspect, the present disclosure provides an anticancer peptide comprise at least 90% sequence identity with an amino acid sequence selected from any of SEQ ID NO: 1- 23. In any embodiment, the amino acid sequence may be selected from SEQ ID NO: 2, SEQ IDNO: 4, and SEQ ID NO: 5, or a sequence having at least 90% sequence identity thereto. In any embodiment, the amino acid sequence may be selected from SEQ ID NO: 18 and SEQ ID NO: 23, or a sequence having at least 90% sequence identity thereto. In any embodiment, the amino acid sequence may comprise SEQ ID NO: 5, or a sequence having at least 90% sequence identity with SEQ ID NO: 5. In any embodiment, the sequence identity may be at least 95%. In any embodiment, the sequence identity may be at least 99%. In any embodiment, the sequence identity may be 100%. In any embodiment, the ammo acid sequence may comprise SEQ ID NO: 4, or a sequence having at least 90% sequence identity7with SEQ ID NO: 4. In any embodiment, the sequence identity may be at least 95%. In any embodiment, the sequence identity may be at least 99%. In any embodiment, the sequence identity may be 100%. In any embodiment, the amino acid sequence may comprise SEQ ID NO: 18, or a sequence having at least 90% sequence identity with SEQ ID NO: 18. In any embodiment, the sequence identity may be at least 95%. In any embodiment, the sequence identity may be at least 99%. In any embodiment, the sequence identity may be 100%. In any embodiment, the amino acid sequence may comprise SEQ ID NO: 23, or a sequence having at least 90% sequence identity with SEQ ID NO: 23. In any embodiment, the sequence identity may be at least 95%. In any embodiment, the sequence identity may be at least 99%. In any embodiment, the sequence identity may be 100%.

[0007] In any embodiment of the anticancer peptide, one or more amino acids of the amino acid sequence may be modified. In any embodiment of the anticancer peptide, one or more amino acids of the amino acid sequence may be phosphorylated, glycosylated, nitrosylated, biotinylated, methylated, acetylated, or lipidated. In any embodiment wherein one or more amino acids of the amino acid sequence is glycosylated, the one or more glycosylated amino acids may be N-glycosylated, O-glycosylated, C-glycosylated, glypiated. or phosphoglycosylated.

[0008] In another aspect, the present disclosure provides a pharmaceutical composition comprising one or more anticancer peptides as described herein. In any embodiment, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, a pharmaceutically acceptable preservative, a pharmaceutically acceptable antioxidant, a pharmaceutically acceptable salt, a pharmaceutically acceptable pH modulator, a pharmaceutically acceptable buffer, or any combination thereof. In any embodiment, the anticancer peptide may comprise at least 99% purity. In any embodiment, the composition may be essentially free of peptides, nucleic acids, and cell fragments, other than the anticancer peptide.

[0009] In another aspect, the present disclosure provides a kit comprising the pharmaceutical composition as described herein; a container; a label; and instructions that provide methods of administering the pharmaceutical composition.

[0010] In still another aspect, the present disclosure provides a method of generating anticancer peptides, the method comprising: contacting mammalian cells with one or more cell growth signaling pathway activators; growing the mammalian cells in culture medium; removing the mammalian cells from the culture medium; adding a protein digestion enzyme to the culture medium; and purifying peptides of about 100 amino acids or less in length from the culture medium.

[0011] In any embodiment of the method, the mammalian cells may be mesenchymal stem cells (MSCs). In any embodiment, contacting mammalian cells with one or more cell growth signaling pathway activators may comprise contacting mammalian cells with two or more, three or more, four or more, or five or more cell growth signaling pathway activators. In any embodiment, the cell growth signaling pathway activators may be selected from Wnt signaling pathway activators. PI3K signaling pathway activators, FGF signaling pathway activators, and Notch signaling pathway activators. In any embodiment, the cell growth signaling pathway activators are selected from cMyc, Oct4, Klf4, and Sox2, or variants, homologs, derivatives, or fragments of any thereof. In any embodiment, the cell growth signaling pathway activators are selected from cMyc, Oct4, Klf4, and Sox2. In any embodiment, the protein digestion enzyme may be trypsin.

[0012] In any embodiment, removing the mammalian cells from the culture medium may comprise one or more rounds of centrifugation. In any embodiment, the centrifugation may be at from about 2,000 to about 4,000 rpm.

[0013] In any embodiment, the method may further comprise filtering the culture medium. In any embodiment, the filtering may be with a 0.22-pm poly ethersulfone membrane.

[0014] In any embodiment, the culture medium may comprise fetal bovine serum. In any embodiment, the culture medium may further comprise one or more antibiotics. In any embodiment, the growing mammalian cells in culture medium may be performed at about 37°C. In any embodiment, the gro ing mammalian cells in culture medium may be performed at about 5% CO2 atmosphere.

[0015] In any embodiment, the purifying may comprise extracting via ion exchange chromatography, gel permeation chromatography, medium-pressure reversed phase chromatography, or high-pressure reversed phase chromatography.

[0016] In any embodiment, the method may further comprise eluting the purified peptides.

[0017] In still another aspect, the present disclosure provides a method of killing cancer cells, the method comprising: contacting the cancer cells with an anti cancer peptide as described herein. In any embodiment, the cancer cells may be human cancer cells. In any embodiment, the cancer cells may be selected from any of: osteosarcoma cells, breast cancer cells, or pancreatic cancer cells. In any embodiment, the cancer cells may be osteosarcoma cells. In any embodiment, the cancer cells may be breast cancer cells. In any embodiment, the cancer cells may be pancreatic cancer cells. In any embodiment, the cancer cells may be pancreatic ductal adenocarcinoma cells.

[0018] In another aspect, the present disclosure provides use of an anticancer peptide as described herein or pharmaceutical composition as described herein or composition as described herein for the manufacture of a medicament for the therapeutic treatment of cancer. In any embodiment of such use, the cancer may be osteosarcoma. In any embodiment of such use, the cancer may be breast cancer. In any embodiment of such use, the cancer may be pancreatic cancer. In any embodiment, the cancer may be pancreatic ductal adenocarcinoma.

[0019] In another aspect, the present disclosure provides a composition comprising a polynucleotide encoding an anticancer peptide as described herein. In any embodiment, the polynucleotide may be encoded in a vector. In any embodiment, the vector may be selected from the group consisting of plasmids, viral vectors, bacteriophages, cosmids, artificial chromosomes, and any combination thereof. In any embodiment, the viral vector may comprise an adeno-associated virus.

[0020] In another aspect, the present disclosure provides a kit comprising the composition comprising a polynucleotide encoding an anti cancer peptide as described herein; a container; a label; and instructions that provide methods of administering the composition.

[0021] In yet another aspect, the present disclosure provides a method of treating a cancer in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of the anticancer peptide described herein or administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein or administering to the subject a therapeutically effective amount of the composition described herein.

[0022] In any embodiment of the method of treating a cancer in a subj ect in need thereof, the subject may be a human. In any embodiment, the cancer may be selected from: osteosarcoma, breast cancer, or pancreatic cancer. In any embodiment, the cancer may beosteosarcoma. In any embodiment, the cancer may be breast cancer. In any embodiment, the cancer may be pancreatic cancer. In any embodiment, the cancer may be pancreatic ductal adenocarcinoma. In any embodiment, the cancer may be metastatic. In any embodiment, the cancer may be incipient. In any embodiment, treating the cancer may reduce a tumor size. In any embodiment, treating the cancer may increase bone volume ratio, increase bone mineral density, increase trabecular number, reduce trabecular separation, or reduce osteoclastogenesis. In any embodiment, treating the cancer may treat cancer-onset diabetes or cancer-related jaundice or both.

[0023] In another aspect, the present disclosure provides a kit comprising an anticancer peptide as described herein; a container; a label; and instructions that provide methods of administering the anticancer peptide.

[0024] In another aspect, the present disclosure provides an anticancer peptide, a pharmaceutical composition, a kit, a method, or a use as substantially described herein.

[0025] In another aspect, the present disclosure provides an anticancer pharmaceutical composition comprising a peptide of SEQ ID NO: 18 or SEQ ID NO: 23. In any embodiment of the anticancer pharmaceutical composition, the peptide may comprise an N-terminus acetyl modification, a C-terminus amino-group modification, or both an N-terminus acetyl modification and a C-terminus amino-group modification. In any embodiment, the composition may further comprise cisplatin, taxol, or both cisplatin and taxol.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The concepts described herein are illustrative by way of example and not by way of limitation in the accompanying figures. For simplicity and clarity of illustration, elements illustrated in the figures are not necessarily drawn to scale. Where considered appropriate, references labels have been repeated among the figures to indicate corresponding or analogous elements.

[0027] Figs. 1A-1E show the anti-tumor effect of P04 on pancreatic ductal adenocarcinoma (PDAC) cell lines. CN = control. The single and double asterisks indicate p < 0.05 and 0.01, respectively, (a) The amino acid sequences of the selected three peptides, P02, P04, and P07; (b) Reduction in MTT-based viability of PANCI, PAN198, and PA03C pancreatic tumor cells in response to 25 pg / ml of P02, P04, and P07; (c) Reduction in scratchbased motility of PANCI cells in response to 25 pg / ml of P04; (d) and (e) Description of what is contained in the second panel.

[0028] Figs. 2A-2E show inhibitory’ effects on PDAC tissue fragments in response to P04. The single and double asterisks indicate p < 0.05 and 0.01, respectively, (a) The illustrationof PDAC tissue fragments assay; (b&c) Reduction in PDAC fragment size in 96 h in response to 25 pg / ml of P04 (n=8). The red image indicates tissue fragments at 0 h, while the green image indicates them at 96h; (d&e) Reduction in PDAC fragment size in 96 h in response to 10 pM of 5-Flu with and without 25 pg / ml of P04 (n=8). The red image indicates tissue fragments at 0 h, while the green image indicates them at 96 h.

[0029] Figs. 3A-3B show inhibitory effects of P04 in combination with Gemcitabine and 5-Flu. CN = control, Gem = Gemcitabine, and 5-Flu = 5-Fluorouracil. (a) Illustration of combinatorial anti-tumor effects of P04, together with Gemcitabine or 5-Flu; (b) Decrease in the levels of p-Src and Snail, as well as an increase in cleaved caspase-3 (c-Cas-3) in PANCI and PAN 198 PDAC cells in response to of 25 pg / ml of P04.

[0030] Figs. 4A-4E show generation of induced tumor-suppressing cells (iTSCs) by the overexpression of ALDOA in mesenchymal stem cells (MSCs). CN = control, pl = plasmid transfection, MSC = mesenchymal stem cell, and CM = conditioned medium. The double asterisk indicates p < 0.01. (a&b) The elevated ALDOA transcript level in patients with PDAC, and lower survival rate for patients with a high ALDOA level in TCGA database; (c-e) Reduction in MTT-based viability . EdU-based proliferation, scratch-based motility, and Transwell® invasion, respectively, by ALDOA-overexpressing MSC-derived conditioned medium.

[0031] Figs. 5A-5C illustrate a predicted interaction between P04 and EGFR protein, (a) Reduction in MTT-based viability of PANCI cells in response to 5 pg / ml of recombinant ALDOA protein; (b) Decrease in the levels of ALDOA and K-Ras in PANC I cells by the application of 25 pg / ml of P04; (c) Predicted interactions between P04 and EGFR by molecular docking using Z-DOCK software.

[0032] Figs. 6A-6D show generation of iTSCs from PBMCs by overexpressing ALDOA. CN = control, pl = plasmid transfection, PBMC = peripheral blood mononuclear cells, and CM = conditioned medium. The single and double asterisks indicate p < 0.05 and 0.01, respectively, (a) Elevated ALDOA level by transfection of ALDOA plasmids; (b) Reduction in MTT-based viability of PANCI and PAN 198 cells by ALDOA-overexpressing PBMC-derived CM; (c&d) Shrinkage of PDAC fragments by ALDOA-overexpressing PBMC-derived CM with and without P04.

[0033] Figs. 7A-7D show generation of iTSCs from PBMCs by the application of low- intensity vibration. CM = conditioned medium, CN = control, and LIV = vertical low-intensity vibration, (a) Reduction in MTT-based viability of PANCI cells by the application of LIV - treated Jurkat cell-derived CM; (b) An elevated level of ALDOA in PBMCs in response to LIV;(c) Reduction in MTT-based viability and scratch-based motility of PANCI cells by LIV -treated PBMC-derived CM; (d) No detectable changes in MTT-based viability and scratch-based motility of MSCs by LIV -treated PBMC-derived CM.

[0034] Figs. 8A-8D show generation of iTSCs from Jurkat cells by LIV at different viscosity. CM = conditioned medium, CN = control, LIV = vertical low-intensity vibration, and cP = centipoise (viscosity unit), (a&b) Reduction in MTT-based viability of PANCI and PAN198 cells by the application of LIV-treated Jurkat-derived CM; (c) Reduction in scratchbased motility of PANCI cells by LIV-treated Jurkat-derived CM; (d) Illustration of a proposed mechanism of the anti-tumor action of ALDOA, P04, and vibration-treated PBMC-derived CM.

[0035] Figs. 9A-9C show suppression of tumorigenic behaviors of PANCI 98 cells in response to ALDOA-overexpressing MSC-derived conditioned medium (CM). CM = conditioned medium, CN = control, MSC = mesenchymal stem cell, ALDOA = aldolase A, and pl = plasmid transfection. The single and double asterisks indicate p < 0.05 and p < 0.01, respectively. The double hash sign indicates p < 0.01 to normal control. (Fig. 9A) Reduction in MTT-based viability and EdU-based proliferation in response to ALDOA-overexpressing MSC-derived CM. (Fig 9B) Reduction in scratch-based motility in response to ALDOA- overexpressing MSC-derived CM. (Fig. 9C) Reduction in transwell-based invasion in response to ALDOA-overexpressing MSC-derived CM.

[0036] Figs. 10A-10F show suppression of tumorigenic behaviors of pancreatic cancer cells and breast cancer cells in response to vibration-treated Jurkat cell-derived conditioned medium (CM). The single and double asterisks indicate p < 0.05 and 0.01 , respectively. CM = conditioned medium, CN = control, LIV = low-intensity vibration, and cP = centi-Poise (viscosity unit) modulated by methylcellulose. The single and double hash signs indicate p < 0.05 and p < 0.01 to normal control, respectively. The single and double asterisks indicate p < 0.05 and p < 0.01 to CM control, respectively. (Figs. 10A&B) Reduction in MTT-based viability of PANCI and PANC198 pancreatic cancer cell lines, respectively, in response to LIV- treated CM. Of note, the CM with the highest viscosity7(20 cP) showed a stronger reduction than the LIV-treated control CM. (Fig. 10C) Reduction in scratch-based motility of PANCI cells in response to LIV-treated CM. (Fig. 10D) Elevated level of phosphorylated Akt (p-Akt) in Jurkat cells by the application of LIV. (Figs. 10E&F) Reduction in MTT-based viability of MDA-MB-231 and MDA-MB-436 breast cancer cell lines, respectively, in response to LIV- treated CM with higher viscosity (20 cP for MDA-MB-231 cells and 5, 10, and 20 cP for MDA- MB-436 cells).

[0037] Figs. 11A-11D show antitumor activity of ACPs P02, P04, and P05. MSC = mesenchymal stem cell, and CN = control. The single and double asterisks indicate p < 0.05 and 0.01, respectively. Figs. 11 A-l IB illustrate reduction of MTT-based viability and scratch-based motility of U2OS, TT2, and MG63 osteosarcoma cells in response to 10 and 50 pg / ml P02, P04, and P05. Figs. 11C-1 ID show no significant effects on MTT-based viability and scratch-based mo-tility of MSCs in response to 10 and 50 pg / ml P02, P04, and P05.

[0038] Figs. 12A-12C show dose-dependent effects of P02. P04. and P05 on cell viability and cell motility. CN = control. The single and double asterisks indicate p <0.05 and 0.01, respectively. Fig. 12A shows effects of P02, P04, and P05 on MTT-based viability of TT2 osteosarcoma cells. Figs. 12B-12C show combined effects of P02, P04, and P05 on MTT-based viability and scratch-based motility of TT2 osteosarcoma cells.

[0039] Figs. 13 A-l 3D show effects of P05 on proliferation, invasion, and gene expression, and its combined effects with the selected chemotherapeutic agents. CN = control, Cis = Cisplatin, and Dox = Doxorubicin. The double asterisk indicates p < 0.01. Figs. 13A-13B show effects of 25 pg / ml P05 on proliferation and Transwell® invasion of TT2 osteosarcoma cells. Fig. 13C shows Decrease in the levels of p-Src, EGFR, and Snail, and an increase in cleaved caspase-3 (c-Cas-3) in TT2 osteosarcoma cells by the application of 25 pg / ml P05. Fig. 13D shows combinatorial anti-tumor effects of P05 with Cisplatin, Doxorubicin, or Taxol.

[0040] Fig. 14A-14E show anti-tumor effects of P02, P04, and P05 on MDA-MB-231 breast cancer cells. CN = control. The single and double asterisks indicate p < 0.05 and 0.01, respectively. Figs. 14A-14D show reduction in MTT-based viability, scratch-based motility. Transwell® invasion, and EdU-based prolifera-tion, respectively. Fig. 14E shows downregulation of oncoproteins such as EGFR, p-Src, and Snail by P05.

[0041] Fig. 15A-15F sho s double-edged role of ALDOA to osteosarcoma and MSCs. CN = control, pl = plasmid transfection, MSC = mesenchymal stem cell, and CM = conditioned medium. The double asterisk indicates p < 0.01. Fig. 5A shows increase in MTT-based viability and the level of EGFR by overexpressing ALDOA in TT2 osteosarcoma cells. Fig. 15B shows overexpression of ALDOA in MSCs and the elevation of EGFR. Figs. 15C-15F show reduction in MTT-based viability, EdU-based proliferation, scratch-based motility, and Transwell® invasion, respectively, by ALDOA-overexpressing MSC-derived conditioned medium.

[0042] Figs. 16A-16E shows possible involvement of EGFR in the action of ALDOA and P05. CN = control. The double asterisk indicates p < 0.01. Figs. 16A-16B show predicted interactions of ALDOA-EGFR and P05-EGFR. Figs. 16C-16D Reciprocal immunoprecipitation of EGFR and ALDOA using TT2 derived cell lysates. Fig. 16E showssuppression of ALDOA-driven reduction in MTT-based viability of TT2 OS cells by silencing EGFR.

[0043] Figs. 17A-17C illustrate P05:s protective effects on bone and the proposed mechanism of P05’s action. CN = control. The double asterisk indicates p < 0.01. Fig. 17A shows reduction in multi -nucleated RANKL-stimulated osteoclasts in response to P05. Fig. 17B shows reduction in EGFR, M-CSF, OPN, IL-1(3. IL6, and p-Src by the administration of 25 pg / mL of P05 in the ex-vivo bone culture. Fig. 17C illustrates a proposed mechanism of the antitumor action of P05.

[0044] Figs. 18A-18B show the effect of P02, P04, and P05 on hFOB cells. CN = control. Figs. 18A-18B show no significant change in the MTT-based viability and scratchbased motility, respectively, in response to the administration of 25 pg / ml of P02, P04. or P05.

[0045] Figs. 19A-19D show the anti- tumor action of P02, P04, and P05 to U2OS and MG63 osteosarcoma cells. CN = control. The single and double asterisks indicate p < 0.05 and 0.01, respectively. Figs. 19A-19B show reduction in scratch-based motility of U2OS and MG63 osteosarcoma cells, respectively, by the application of 50 pg / ml of P02. P04, and P05. Fig. 19C shows reduction in EdU-based proliferation of MG63 cells by 25 pg / ml of P05. Fig. 19D shows downregulation of EGFR, p-Src, and Snail in MG63 cells by 25 pg / ml of P05.

[0046] Figs. 20A-20D show the tumor-suppressing role of ALDOA-overexpressing MSCs. CN = control, p = plasmid transfection, MSC = mesenchymal stem cell, and CM = conditioned medium. The double asterisk indicates p < 0.01. Fig. 20A shows the contrasting role of ALDOA. The overexpression of ALDOA in MG63 OS cells elevated their MTT-based viability. However, ALDOA-overexpressing MSC-derived conditioned medium reduced the MTT-based viability of MG63 OS cells. Figs. 20B-20D show reduction in scratch-based motility, EdU-based proliferation, and Transwell® invasion, respectively, by ALDOA- overexpressing MSC-derived conditioned medium.

[0047] Figs. 21A-21C show the predicted network of the ALDOA-interacting protein candidates and the effect of P02, P04, and P05 on MC3T3-E1 osteoblast cells. Fig. 21 A shows a prediction of ALDOA-interaction protein candidates based on network. Fig. 2 IB shows no significant change in the MTT-based viability of MC3T3-E1 cells in response to the administration of 25 pg / ml of P02, P04, or P05 peptides. Fig. 21 C shows slight upregulation of type I collagen (COLI) and alkaline phosphatase (ALP) in response to the administration of 25 pg / ml of P05 peptides.

[0048] Figs. 22a-22g show the anti-tumor effect of the peptide P18 on breast cancer cells. CN = control. The single and double asterisks indicate p < 0.05 and 0.01, respectively.Fig. 22a shows reduction in MTT-based viability of MDA-MB-231, MDA-MB-436, and MCF7 breast cancer cells in response to 5, 10, 15, and 25 pg / ml of Pl 8. Fig. 22b shows reduction in scratch-based motility of MDA-MB-231 cells in response to 25 pg / ml of Pl 8. Figs. 22C and 22d Inhibitory effects of 25 pg / ml of Pl 8 on EdU-based proliferation (Fig. 22c) and Transwell® invasion (Fig. 22d) of MDA-MB-231 cells. Fig. 22e shows reduction in scratch-based motility of MDA-MB-436 cells in response to 25 pg / ml of P18. Figs. 22f and 22g show inhibitory effects of 25 pg / ml of P18 on EdU-based proliferation (Fig. 22f) and Transwell® invasion (Fig. 22g) of MDA-MB-436 cells.

[0049] Figs. 23a-23f show inhibitory effects of P18 on pancreatic ductal adenocarcinoma (PDAC), osteosarcoma, and breast tumor cells. CN = control. The single and double asterisks indicate p < 0.05 and 0.01, respectively. (Fig. 23a) Reduction in MTT-based viability of PANCI and PANC198 PDAC cells in response to 5, 10, 15, and 25 pg / ml of P18. (Fig. 23b) Reduction in MTT-based viability of TT2 OS cells in response to 5, 10, 15, and 25 pg / ml of P18. (Figs. 23c&d) Inhibitory effects of 25 pg / ml of P18 on MTT-based viability' (Fig. 23c) and scratch-based motility (Fig. 23d) of 4T1.2 mammary tumor cells. (Figs. 23e&f) No significant effects on MTT-based viability (Fig. 23e) and scratch-based motility (Fig. 23f) of MSCs in response to 25 pg / ml of Pl 8.

[0050] Figs. 24a-24c show inhibitory' effects of Pl 8 on MDA-MB-231 breast cancer cells and breast cancer tissue fragments in combination with Cisplatin and Taxol. CN = control, and Cis = Cisplatin. The single and double asterisks indicate p < 0.05 and 0.01, respectively, while the single hash mark indicates p < 0.05 for the comparison within the Cisplatin and Taxol treatments. (Fig. 24a) Additive anti-tumor effects of Pl 8, with Cisplatin or Taxol. (Fig. 24b) Reduction in breast cancer tissue fragment size in 96 h in response to 25 pg / ml of P18 (n = 6). The red images (the bottom of the top row of Fig. 24b) indicate tissue fragments at 0 h. while the green images (in the middle row and bottow row of Fig. 24b) indicate them at 96 h. (Fig. 24c) Reduction in breast cancer fragment size in 96 h in response to 5 pM of Cisplatin or Taxol with and without 25 pg / ml of P18 (n = 6).

[0051] Figs. 25a-25c show anti-tumor effects of P04, P05, P14, and P20 with the combination of P18 on MDA-MB-231 and MDA-MB-436 breast cancer cells. CN = control. The double asterisk indicates p < 0.01. (Fig. 25a) Effects of P04, P05, P18 and their combinatorial usage on MTT-based viability' of MDA-MB-231 and MDA-MB-436 breast cancer cells. (Fig. 25b) Decrease in the levels of p-Src, and Snail, as well as an increase in cleaved caspase 3 (c-Cas-3) in MDA-MB-231 (left) and MDA-MB-436 (right) breast cancercells by the application of 25 pg / ml Pl 8. (Fig. 25c) Significant reduction in multi-nucleated RANKL-stimulated osteoclasts in response to Pl 8.

[0052] Figs. 26a-26d show P 18 and P18-consisting ARHGDIA with their possible binding partners. (Fig. 26a) The lowered survival rate for patients with a high ARHGDIA transcript level in the National Cancer Institute's The Cancer Genome Atlas (TCGA) database (Fig. 26b) Reduction in MTT-based viability’ of MDA-MB-231 cells in response to 5 pg / mL of recombinant ARHGDIA protein. (Fig. 26c) Prediction of ARHGDIA-interacting proteins including GTPases such as Rael, RhoA, and Cdc42. (Fig. 26d) Slight increase in the levels of Cdc42 and RhoA in MDA-MB-231 (left) and MDA-MB-436 (right) breast cancer cells by the application of 25 pg / ml Pl 8.

[0053] Figs. 27a-27c show effects of P18 on GTPase activities of Rael. RhoA. and Cdc42 in MDA-MB-231 breast cancer cells. The FRET-based GTPase activity is color-coded in individual cells, in which data were normalized and compared to the control group (CN). The scale bars indicate 50 pm. *** p < 0.001, **** p < 0.0001. (Fig. 27a) No detectable effect of Pl 8 on Rael GTPase activity. (Figs. 27b&c) Suppression of GTPase activities of RhoA (Fig. 27b) and Cdc42 (Fig. 27c), respectively, in response to Pl 8.

[0054] Figs. 28a-28e show evaluation of amino acid residues in Pl 8 through molecular docking analysis and modification of both ends of Pl 8. CN = control. (Fig. 28a) Predicted interactions between Cdc42 and ARHGDIA (Cdc42 in blue (the more compact element, shown on the right side of the modeled duplex), and ARHGDIA in yellow (the larger, less compact element, shown on the left side of the modeled duplex). (Fig. 28b) Predicted interactions between Cdc42 and P18 (Cdc42 in blue (the larger element), and P18 in yellow (the small element show n on the upper left of the modeled duplex). (Figs. 28c&d) MTT-based viability of MDA-MB-231 and MDA-MB-436 (Fig. 28c). and MCF7 (Fig. 28d) breast cancer cells in response to two P18 modified analogs, (e) The schematic diagram of P18 modified analogs.

[0055] Figs. 29a-29b show bone protective effects and the proposed mechanism of P18’s action. (Fig. 29a) Reduction in p-Src and Snail, and an increase in cleaved caspase 3 (c- Cas3) and alkaline phosphatase (ALP) by the daily administration of 25 pg / rnL of Pl 8 in the ex-vivo bone culture. The levels of RhoA, p-Akt, and RANKL were slightly reduced. However, the level of Cdc42 w as slightly elevated. (Fig. 29b) Proposed mechanism of the anti-tumor and bone-protective action of Pl 8.

[0056] Figs. 30a-30b show inhibitory effects of Pl 8 on triple-negative breast cancer tissue fragments in combination with Cisplatin and Taxol after 48h treatment. CN = control. The single and double asterisks indicate p < 0.05 and 0.01, respectively, while the single hashmark indicates p < 0.05 for the comparison within Cisplatin and Taxol treatments. (Fig. 30a) Reduction in breast cancer tissue fragment size in response to 25 pg / ml of P18 (n = 6). (Fig. 30b) Reduction in breast cancer tissue fragment size in response to 5 pM of Cisplatin (left) or Taxol (right) with and without 25 pg / ml of P18 (n = 6).

[0057] Figs. 31a-31b show altered effects of P18 analogs on MDA-MB-231 and MDA- MB-436 breast cancer cells as well as TT2 osteosarcoma cells. CN = control, P18-N5 = P18 analog shifted to the N-terminus by 5 amino acids, and P18-C5 = Pl 8 analog shifted to the C- terminus by 5 amino acids. The double asterisk indicates p < 0.01. (Fig. 31a) MTT-based viability of MDA-MB-231 (left) and MDA-MB-436 (right) breast cancer lines. (Fig. 31b) MTT- based viability of TT2 osteosarcoma cells.

[0058] Figs. 32a-32d show evaluation of amino acid residues in Pl 8 through Alanine replacement. CN = control. (Figs. 32a&b) MTT-based viability of MDA-MB-231 (Fig. 32a) and MDA-MB-436 (Fig. 32b) breast cancer lines in response to eleven Pl 8 analogs. These analogs are designed by a replacement of each of the amino acids in P18, (Figs. 32c&d) MTT- based viability of TT2 (Fig. 32c) cells and PANCI (Fig. 32d) pancreatic cancer cells in response to eleven P18 analogs. These analogs are designed by a replacement of each of the amino acids in Pl 8, #142 to #152, by alanine.

[0059] Figs. 33a-33b show evaluation of anti-tumor effect of P18 analogs that have both ends modified. CN = control. (Fig. 33a&b) MTT-based viability of 4T1.2 breast cancer cells and TT2 OS cells (Fig. 33a); and PANCI and PAN198 PDAC cells (Fig. 33b) in response to the two Pl 8 analogs.

[0060] Figs. 34a-34d show evaluation of the anti -tumor effect of Pl 8 analogs with a single C-terminal modification. CN = control. (Figs. 34a&b) MTT-based viability of MDA- MB-231 and MDA-MB-436 (Fig. 34a); and 4T1.2, and MCF7 (Fig. 34b) breast cancer lines in response to the C-terminal modified P18 analog. (Figs. 34c&d) MTT-based viability of PANCI and PAN198 PDAC cells (Fig. 34c); and TT2 OS (Fig. 34d) cell lines in response to C-terminal modified Pl 8 analog (sequence shown on the lower right).DETAILED DESCRIPTION

[0061] The subject matter described herein relates to anti-cancer peptides, methods of generating such peptides, and methods of using such peptides for killing cancer cells and / or for treatment of cancer in a subject in need thereof.

[0062] Although the concepts of the present disclosure are susceptible to various modifications and alternative forms, specific embodiments have been shown by way of examplein the drawings and will be described herein in detail. It should be understood, however, that there is no intent to limit the concepts of the present disclosure to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives consistent with the present disclosure and the appended claims.

[0063] References in the specification to “one embodiment,” “an embodiment,” “an illustrative embodiment,” etc., indicate that the embodiment described may include a particular feature, structure, or characteristic, but every embodiment may or may not necessarily include that particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. It should be further appreciated that although reference to a “preferred” component or feature may indicate the desirability of a particular component or feature with respect to an embodiment, the disclosure is not so limiting with respect to other embodiments, which may omit such a component or feature. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to implement such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described. Additionally, it should be appreciated that items included in a list in the form of “at least one of A, B, and C” can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C). Similarly, items listed in the form of “at least one of A, B, or C” can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C). Further, with respect to the claims, the use of words and phrases such as “a.” “an,” “at least one,” and / or “at least one portion” should not be interpreted so as to be limiting to only one such element unless specifically stated to the contrary, and the use of phrases such as “at least a portion” and / or “a portion” should be interpreted as encompassing both embodiments including only a portion of such element and embodiments including the entirety of such element unless specifically stated to the contrary.

[0064] In the drawings, some structural or method features may be shown in specific arrangements and / or orderings. However, it should be appreciated that such specific arrangements and / or orderings may not be required. Rather, in some embodiments, such features may be arranged in a different manner and / or order than shown in the illustrative figures unless indicated to the contrary. Additionally, the inclusion of a structural or method feature in a particular figure is not meant to imply that such feature is required in all embodiments and, in some embodiments, may not be included or may be combined with other features.

[0065] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, applications, published applications and other publicationsreferred to herein are incorporated by reference in their entireties. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in a patent, application, or other publication that is herein incorporated by reference, the definition set forth in this section prevails over the definition incorporated herein by reference.

[0066] The singular forms “a,” “an,"’ and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. The terms “including,” “containing,” and “comprising” are used in their open, non-limiting sense.

[0067] To provide a more concise description, some of the quantitative expressions given herein are not qualified with the term “about.” It is understood that, whether the term “about” is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and / or measurement conditions for such given value.

[0068] Certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the chemical groups represented by the variables are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace compounds that are stable compounds (i.e., compounds that can be isolated, characterized, and tested for biological activity). In addition, all subcombinations of the chemical groups listed in the embodiments describing such variables are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such subcombination of chemical groups was individually and explicitly disclosed herein.

[0069] The present disclosure provides anticancer peptides (ACPs). methods of making such ACPs, and method of using such ACPs to kill cancer cells and to treat a subject in need thereof. The ACPs may be given to patients, and may inhibit cancer progression or reverse cancer progression or both. The ACPs are effective to inhibit growth and migration of cancer cells. Without being limited by theory, it is believed that the ACPs bind to molecular targets that are highly expressed in cancer cells, and inhibit oncogenic action of the target molecules.

[0070] As used herein “anticancer peptide” refers to a peptide that possess anti-tumor activity, such as the ability to shrink a tumor or even completely eliminate a tumor, regardless the mechanism and / or whether the mechanism is unknown. Anticancer peptides (ACPs) have recently emerged as a promising therapeutic choice for many types of cancer, potentially, because of their high penetration and specificity with fewer side effects. Peptides are bioactive molecules comprising a polymer chain of amino acids, but which are generally smaller than proteins (which also comprise a polymer chain of amino acids). Generally, a peptide may be 2 to about 100 amino acids. Peptide chains are usually linear, but may be branched or circularized or both branched and circularized. Further, one or more amino acids may have chemical modifications, such as hybridizing to a sugar moiety, forming a glycopeptide. Compared to tumor-suppressing proteins, peptides are generally easier to synthesize and are considered less likely to cause drug resistance, although they may have drawbacks in their stability with a short half-life. Several computational approaches have been developed to predict the anticancer activities of ACP candidates, but it is still a challenge to design effective ACPs since a variety of mechanisms of anti cancer action are considered.

[0071] “Bone mineral density” refers to the inorganic mineral content in bone.

[0072] “Bone volume ratio” refers to the ratio of the segmented bone volume to the total volume.

[0073] “Cell growth signaling pathway activator” refers to any substance that enhances, or promotes or activates non-cancerous or cancerous mammalian cell growth and / or cell proliferation activity and / or cell migration activity’. Mammalian cell growth signaling pathways include, but are not limited to, highly conserved pathways such as the Wnt signaling pathway, the PI3K signaling pathway, the Fibroblast Growth Factor (FGF) signaling pathway, and the Notch signaling pathway. Cell growth signaling pathway activators useful in the embodiments include small molecules, proteins, fusion proteins, and / or nucleic acids. In embodiments, cell growth signaling pathways include, but are not limited to the Wnt signaling pathway, the OCT3 / 4 signaling pathway, the PI3K signaling pathway, the Ras-ERK signaling pathway, the Fibroblast Growth Factor (FGF) signaling pathway, the Notch signaling pathway, the c-Myc signaling pathway, and the Epithelial Mesenchymal Transition (EMT) signaling pathway.

[0074] “Cell growth signaling pathway inhibitors” refers to any substance that diminishes or inhibits or inactivates non-cancerous or cancerous mammalian cell growth and / or cell proliferation activity. Cancerous cells are not treated with cell growth signaling pathway inhibitors in embodiments.

[0075] “Wnt signaling pathway'’ denotes a signaling pathway that may be divided in two pathways: the canonical Wnt / beta catenin signaling pathway and the "Wnt / PCP signaling pathway. Canonical Wnt / beta catenin signaling pathway” or Wnt / PCP signaling pathway denotes a network of proteins and other bioactive molecules (lipids, ions, sugars . . . ) best known for their roles in embryogenesis and cancer, but also involved in normal physiological processes in adult animals. The canonical Wnt / beta catenin signaling pathway is characterized by a Wnt dependant inhibition of glycogen synthase kinase 3[3 (GSK.-3P), leading to a subsequent stabilization of P-catenin, which then translocates to the nucleus to act as a transcription factor. The Wnt / PCP signaling pathway does not involve GSK-3P or P-catenin, and comprises several signaling branches including Calcium dependant signaling, Planar Cell Polarity (PCP) molecules, small GTPases and C-Jun N-terminal kinases (JNK) signaling. These pathways are well known to those skilled in the art.

[0076] “Wnt signaling pathway activator” refers to a substance that enhances or promotes or activates a Wnt signaling activity'. For example, for the canonical Wnt / p-catenin signaling pathway, this activity can be measured by Wnt reporter activity using established multimers of LEF / TCF binding sites reporters, and / or inhibition of GSK.-3P, and / or activation of canonical Wnt target genes such as T, Tbx6, Msgnl, or Axin2. An activation of a Wnt signaling activity' may therefore be assessed as being an increase of a Wnt of Msgnl reporter activity as identified above. The increase may be of at least 1%, 5% 10%, 15%, 20%, 25%, 30%, 35%. 40%. 45%. 50%. 55%. 60%. 65%. 70%. 75%. 80%, 85%, 90%, 95%, 100% or more. Wnt signaling pathway activators are known to those skilled in the art. Small molecule Wnt signaling pathway activators include, but are not limited to, BML-284, CHIR99021, and Wnt pathway activator 1.

[0077] “Epithelial-mesenchymal transition (EMT) pathway” refers to signaling pathways that relate to biologic processes that allow a normal or cancer cell to undergo multiple biochemical changes, thereby enabling it to assume a mesenchymal cell phenotype, e.g., enhanced migratory capacity, invasiveness, elevated resistance to apoptosis, and greatly increased production of ECM components. Classes of molecules that change in expression, distribution, and / or function during EMT, and that are causally involved, include growth factors (e.g., transforming growth factor-P (TGF- ), Wnts), EGF, HGF, transcription factors (e.g.. Snail, SMAD, LEF, and nuclear P-catenin), molecules of the cell-to-cell adhesion axis (cadherins, catenins), cytoskeletal modulators (Rho family), and extracellular proteases (matrix metalloproteinases, plasminogen activators).

[0078] “EMT pathway activator” refers to a substance that enhances or promotes or activates a EMT pathway or activity.

[0079] “PI3K / Akt signaling pathway activator” refers to a substance that enhances or promotes or activates a PI3K / Akt signaling activity. Small molecule PI3K / Akt signaling pathway activators include, but are not limited to YS-49 and SC79.

[0080] “Notch signaling pathway activator” refers to a substance that enhances or promotes or activates a Notch signaling activity. Small molecule Notch signaling pathway activators include, but are not limited to, resveratrol. Small molecule FGF signaling pathway activators, small molecule OCT3 / 4 signaling pathway activators, small molecule c-Myc signaling pathway activators, small molecule Ras-ERK signaling pathway activators, and small molecule EMT signaling pathway activators are known to those skilled in the art.

[0081] Small molecule Oct4 signaling pathway activators include, but are not limited to Oct4 activating compound 2 (OAC2).

[0082] “Cancer” or “tumor” or “cancerous” are well known in the art and refer to the presence, e.g., in a subject, of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, decreased cell death / apoptosis, and certain characteristic morphological features. ’’Cancer” refers to all types of cancer or neoplasm or malignant tumors found in humans, including, but not limited to: leukemias, lymphomas, melanomas, carcinomas and sarcomas. “Cancer,” “neoplasm,” and “tumor.” are used interchangeably and in either the singular or plural form, refer to cells that have undergone a malignant transformation that makes them pathological to the host organism. Primary cancer cells (that is, cells obtained from near the site of malignant transformation) can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of a cancer cell includes not only a primary cancer cell, but also cancer stem cells, as well as cancer progenitor cells or any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells. In certain embodiments, the cancer is a blood tumor (i.e., a non-solid tumor). In some embodiments, the cancer is lymphoid neoplasm diffuse large B-cell lymphoma, cholangiocarcinoma. uterine carcinosarcoma, kidney chromophobe, uveal melanoma, mesothelioma, adrenocortical carcinoma, thymoma, acute myeloid leukemia, testicular germ cell tumor, rectum adenocarcinoma, pancreatic adenocarcinoma, phenochromocytoma and paraganglioma, esophageal carcinoma, sarcoma, kidney renal papillary cell carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, kidney renal clear cell carcinoma, liverhepatocellular carcinoma, glioblastoma multiforme, bladder urothelial carcinoma, colon adenocarcinoma, stomach adenocarcinoma, ovarian serous cystadenocarcinoma, skin cutaneous melanoma, prostate adenocarcinoma, thyroid carcinoma, lung squamous cell carcinoma, head and neck squamous cell carcinoma, brain lower grade glioma, uterine corpus endometrial carcinoma, lung adenocarcinoma, or breast invasive carcinoma. A “solid tumor” is a tumor that is detectable on the basis of tumor mass; e.g.. by procedures such as CAT scan, MR imaging, X-ray, ultrasound or palpation, and / or which is detectable because of the expression of one or more cancer-specific antigens in a sample obtainable from a patient. The tumor does not need to have measurable dimensions.

[0083] Most cancers fall within three broad histological classifications: carcinomas, which are the predominant cancers and are cancers of epithelial cells or cells covering the external or internal surfaces of organs, glands, or other body structures (e.g., skin, uterus, lung, breast, prostate, stomach, bowel), and which tend to metastasize; sarcomas, which are derived from connective or supportive tissue (e.g., bone, cartilage, tendons, ligaments, fat, muscle); and hematologic tumors, which are derived from bone marrow and lymphatic tissue. Carcinomas may be adenocarcinomas (which generally develop in organs or glands capable of secretion, such as breast, lung, colon, prostate or bladder) or may be squamous cell carcinomas (which originate in the squamous epithelium and generally develop in most areas of the body). Sarcomas may be osteosarcomas or osteogenic sarcomas (bone), chondrosarcomas (cartilage), leiomyosarcomas (smooth muscle), rhabdomyosarcomas (skeletal muscle), mesothelial sarcomas or mesotheliomas (membranous lining of body cavities), fibrosarcomas (fibrous tissue), angiosarcomas or hemangioendotheliomas (blood vessels), liposarcomas (adipose tissue), gliomas or astrocytomas (neurogenic connective tissue found in the brain), myxosarcomas (primitive embryonic connective tissue), or mesenchymous or mixed mesodermal tumors (mixed connective tissue types). Hematologic tumors may be myelomas, which originate in the plasma cells of bone marrow; leukemias which may be “liquid cancers” and are cancers of the bone marrow and may be myelogenous or granulocytic leukemia (myeloid and granulocytic white blood cells), lymphatic, lymphocytic, or lymphoblastic leukemias (lymphoid and lymphocytic blood cells) or polycythemia vera or erythremia (various blood cell products, but with red cells predominating); or lymphomas, which may be solid tumors and which develop in the glands or nodes of the lymphatic system, and which may be Hodgkin or Non-Hodgkin lymphomas. In addition, mixed ty pe cancers, such as adenosquamous carcinomas, mixed mesodermal tumors, carcinosarcomas, or teratocarcinomas also exist.

[0084] Cancers may also be named based on the organ in which they originate i.e., the “primary site,” for example, cancer of the breast, brain, lung, liver, skin, prostate, testicle, bladder, colon and rectum, cervix, uterus, etc. This naming persists even if the cancer metastasizes to another part of the body that is different from the primary site. In embodiments disclosed herein, treatment is directed to the site of the cancer, not ty pe of cancer, so that a cancer of any type that is situated in the lung, for example, would be treated on the basis of this localization in the lung.

[0085] For example, cancers include, but are not limited to, mesothelioma, leukemias and lymphomas such as cutaneous T-cell lymphomas (CTCL), noncutaneous peripheral T-cell lymphomas, lymphomas associated with human T-cell lymphotrophic virus (HTLV) such as adult T-cell leukemia / lymphoma (ATLL). B-cell lymphoma, acute nonlymphocytic leukemias, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, lymphomas, and multiple myeloma, non-Hodgkin lymphoma, acute lymphatic leukemia (ALL), chronic lymphatic leukemia (CLL), Hodgkin's lymphoma, Burkitt lymphoma, adult T-cell leukemia lymphoma, acute-myeloid leukemia (AML), chronic myeloid leukemia (CML), or hepatocellular carcinoma. Further examples include myelodisplastic syndrome, childhood solid tumors such as brain tumors, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, and soft-tissue sarcomas, common solid tumors of adults such as head and neck cancers (e.g., oral, laryngeal, nasopharyngeal and esophageal), genitourinary cancers (e.g., prostate, bladder, renal, uterine, ovarian, testicular), lung cancer (e.g., small-cell and non-small-cell), breast cancer, pancreatic cancer, melanoma and other skin cancers, stomach cancer, brain tumors, tumors related to Gorlin's syndrome (e.g., medulloblastoma, meningioma, etc.), and liver cancer. Additional exemplary' forms of cancer which may be treated by the subject compounds include, but are not limited to, cancer of skeletal or smooth muscle, stomach cancer, cancer of the small intestine, rectum carcinoma, cancer of the salivary gland, endometrial cancer, adrenal cancer, anal cancer, rectal cancer, parathyroid cancer, and pituitary cancer.

[0086] Additional cancers and mammalian cancerous cells that are relevant in some embodiments are, for example, colon carcinoma, familial adenomatous polyposis carcinoma and hereditary non-polyposis colorectal cancer, or melanoma. Further, cancers include, but are not limited to, labial carcinoma, larynx carcinoma, hypopharynx carcinoma, tongue carcinoma, salivary' gland carcinoma, gastric carcinoma, adenocarcinoma, thyroid cancer (medullary' and papillary thyroid carcinoma), renal carcinoma, kidney parenchyma carcinoma, cervix carcinoma, uterine corpus carcinoma, endometrium carcinoma, chorion carcinoma, testis carcinoma, urinary’ carcinoma, melanoma, brain tumors such as glioblastoma, astrocytoma.meningioma, medulloblastoma and peripheral neuroectodermal tumors, gall bladder carcinoma, bronchial carcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma, choroidea melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma, and plasmocytoma.

[0087] Osteosarcoma (OS) refers to a primary bone cancer that affects mostly children and young adults. Its common site of induction is the lower limb, and the standard-of-care treatment includes chemotherapy as well as surgical removal. Popular “MAP7’ chemotherapy with three chemotherapeutic agents, including methotrexate, Adriamycin®, and Platinol® (i.e., methotrexate, doxorubicin, and cisplatin), is effective in inhibiting OS’s progression and recurrence. However, advanced OS tends to metastasize, most often to the lung, and it is difficult to treat metastasized OS. The development of effective treatment, as well as early detection, is essential for improving the survival of patients with advanced OS.

[0088] Pancreatic cancer refers to cancers that originate in any tissue that comprises a pancreas. Pancreatic ductal adenocarcinoma (PDAC) refers to a class of cancerous tumor of the pancreas originating from cells that had the capability to form ductal lining and / or originated from ductal lining of the pancreas. A PDAC cell may be found within the pancreas, or found within any organ as a metastasized cell, or found within the blood stream and / or lymphatic system. PDACs typically start in the part of the pancreas that produces digestive enzymes. Early disease histology presents as several distinct subtypes of PDAC precursor lesions, including (but not necessarily limited to) pancreatic intraepithelial neoplasia (PanIN), intraductal papillary’ mucinous neoplasm (TPMN), and mucinous cystic neoplasm (MCN).

[0089] “Triple negative breast cancer (TNBC)” refers to any breast cancer that does not express the genes for estrogen receptor (ER), progesterone receptor (PR) and Her2 / neu. The term includes primary epithelial TNBCs, as well as TNBC that involved with other tumors. The cancer can include a triple negative carcinoma of the breast, ductal carcinoma of the breast, lobular carcinoma of the breast, undifferentiated carcinoma of the breast, cystosarcoma phyllodes of the breast, angiosarcoma of the breast, and primary lymphoma of the breast. TNBC can also include any stage of triple negative breast cancer, and can include breast neoplasms having histologic and ultrastructual heterogeneity (e.g.. mixed cell types).

[0090] “Cell” refers to the basic structural and functional unit of a living organism. In higher organisms, e.g., animals, cells having similar structure and function generally aggregate into “tissues” that perform particular functions. Thus, a tissue includes a collection of similar cells and surrounding intercellular substances, e.g., epithelial tissue, connective tissue, muscle, nerve. An “organ” is a fully differentiated structural and functional unit in a higher organismthat may be composed of different types of tissues and is specialized for some particular function, e.g., kidney, heart, brain, liver, etc. Accordingly, by “specific organ, tissue, or cell” is meant herein to include any particular organ, and to include the cells and tissues found in that organ.

[0091] “Chemotherapeutic agent” refers to a drug used for the treatment of cancer. Chemotherapeutic agents include, but are not limited to, small molecules, hormones and hormone analogs, and biologies (e.g., antibodies, peptide drugs, nucleic acid drugs). In certain embodiments, chemotherapy does not include hormones and hormone analogs.

[0092] “Cancer that is resistant to one or more chemotherapeutic agents” is a cancer that does not respond, or ceases to respond to treatment with a chemotherapeutic regimen, i.e., does not achieve at least stable disease (i.e.. stable disease, partial response, or complete response) in the target lesion either during or after completion of the chemotherapeutic regimen. Resistance to one or more chemotherapeutic agents results in, e.g., tumor grow th, increased tumor burden, and / or tumor metastasis.

[0093] “Combination therapy” includes the administration of the subject compositions / proteins in further combination with other biologically active ingredients (such as, but not limited to, a second and different anti-cancer agent) and non-drug therapies (such as, but not limited to, surgery' or radiation treatment). For instance, the compositions / proteins of some embodiments can be used in combination with other pharmaceutically active compounds, for instance compounds that are able to enhance the effect of the compositions / proteins of some embodiments. The compositions / proteins of some embodiments can be administered simultaneously (as a single preparation or separate preparation) or sequentially to the other drug therapy. In general, a combination therapy envisions administration of a conditioned media / protein / peptide and one or more drugs during a single cycle or course of therapy. In an embodiment, the administered biologically active ingredient is not a cell-growth signaling pathway activator.

[0094] “Conditioned medium” (CM) refers to a liquid nutrient medium that has been in contact with and exposed to cultured non-cancerous cells, or cultured cancerous cells, or cultured mixture of cancerous and non-cancer cells, wherein the cells produce metabolites, peptides, and proteins that enter the media, thus bestowing upon the media a therapeutic activity. In a non-limiting example, CM may be produced by contacting cancerous cells, non-cancerous cells, or a mixture of cancerous and non-cancerous cells, with one or more cell growth signaling pathway activators to generate pre-treated cells; culturing the pre-treated cells in a mammalian cell culture medium for a period of time sufficient to condition the medium; removing the pre-treated cells from the culture medium; and collecting the conditioned medium. Conditioned medium compositions, methods of making conditioned medium, and methods of using conditioned medium are described in International Publication No. WO2022 / 164786 Al to Yokota and International Publication No. WO2022 / 164788 A2 to Yokota, each of which are incorporated by reference herein in their entireties.

[0095] “Disease-free survival” refers to living free of the cancer being monitored. For example, if differential gene expression is used to diagnose or monitor breast cancer, disease- free survival would mean free from detectable breast cancer.

[0096] “Event-free survival” refers to living without the occurrence of a particular group of defined events (for example progression of cancer) after a particular action (e.g., treatment).

[0097] “Mammalian cell culture medium” and “culture medium” (or simply “medium”) refer to a nutrient solution used for growing mammalian cells that typically provides at least one component from one or more of the following categories: (1) salts (e.g., sodium, potassium, magnesium, calcium, etc.) contributing to the osmolality7of the medium; (2) an energy7source, usually in the form of a carbohydrate such as glucose; (3) all essential amino acids, and usually the basic set of twenty amino acids; (4) vitamins and / or other organic compounds required at low concentrations; and (5) trace elements, where trace elements are defined as inorganic compounds that are ty pically required at very7low concentrations, usually in the micromolar range. The nutrient solution may optionally be supplemented with one or more of the components from any of the following categories: (a) animal serum; (b) hormones and other growth factors such as, for example, insulin, transferrin, and epidermal growth factor; and (c) hydrolysates of plant, yeast, and / or tissues, including protein hydrolysates thereof. Selection of the most appropriate culture medium is within the skill of those in the art.

[0098] “Fusion molecule” and “fusion protein” refer interchangeably to a biologically7active polypeptide and an effector molecule covalently linked (i.e., fused) by recombinant, chemical or other suitable method. If desired, the fusion molecule can be fused at one or several sites through a peptide linker sequence. Alternatively, the peptide linker may be used to assist in construction of the fusion molecule. In embodiments, fusion molecules are fusion proteins. Generally fusion molecules also can be comprised of conjugate molecules.

[0099] “Increased” and grammatical equivalents (including “higher,” “bigger,” etc.) when in reference to the expression of any characteristic in a first subject relative to a second subject, mean that the quantity and / or magnitude of the characteristic in the first subject is lower than in the second subject by any amount that is recognized as clinically relevant by any7medically trained personnel. In one embodiment, the quantity and / or magnitude of thecharacteristic in the first subject is at least 10% greater than, at least 25% greater than, at least 50% greater than, at least 75% greater than, and / or at least 90% greater than the quantity and / or magnitude of the characteristic in the second subject. In embodiments, either the first or second subject may be treated and the other of the first or second subject may be untreated. In embodiments, the first subject is untreated and the second subject is treated.

[0100] “Likelihood of reappearance'’ refers to the probability of tumor reappearance or metastasis in a subject subsequent to diagnosis of cancer.

[0101] “Likelihood of recovery” refers to the probability of disappearance of tumor or lack of tumor reappearance resulting in the recovery of the subject subsequent to diagnosis of cancer.

[0102] “Metastasis” is well known to one of skill in the art and refers to the growth of a cancerous tumor in an organ or body part, which is not directly connected to the organ of the original cancerous tumor.

[0103] “Nucleic acids” and “nucleic acid sequences” refer to oligonucleotide, nucleotide, polynucleotide, or any fragment of any of these; and include DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA) of genomic or synthetic origin which may be single-stranded or double-stranded; and can be a sense or antisense strand, or a peptide nucleic acid (PNA), or any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g., double stranded iRNAs, e.g.. iRNPs), nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides.

[0104] “Non-cancerous cells” refers to normal cells. Normal cells can be readily distinguished from primary’ cancer cells and metastatic cancer cells by well-established techniques, particularly histological examination.

[0105] “Selective toxicity” is the propensity of an anti-tumor agent or conditioned medium to affect tumor cells in preference to other healthy cells. In some embodiments, the pharmaceutical compositions and conditioned media are selectively toxic towards tumor cells. In other embodiments, the pharmaceutical compositions and conditioned media are not selectively toxic towards tumor cells and affect cancer cells and healthy cells.

[0106] “Overall survival” refers to the fate of a subject after diagnosis, despite the possibility that the cause of death in a subj ect is not directly due to the effects of the cancer.

[0107] “Pharmaceutically acceptable” and “pharmacologically acceptable” refer to compounds and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.

[0108] In embodiments, the compositions may be administered to patients in a pharmaceutical composition comprising the conditioned medium (CM) along with a pharmaceutically acceptable carrier. The earner may be any solvent, diluent, liquid or solid vehicle that is pharmaceutically acceptable and typically used in formulating compositions. Guidance concerning the making of pharmaceutical formulations can be obtained from standard works in the art (see, e.g., Remington's Pharmaceutical Sciences, 16th edition, E. W. Martin, Easton. Pa. (1980)). In addition, pharmaceutical compositions may contain any of the excipients that are commonly used in the art. Examples of carriers or excipients that may be present include, but are not limited to, sugars (e.g., lactose, glucose and sucrose); starches, such as com starch or potato starch; cellulose and its derivatives (e.g., sodium carboxymethyl cellulose, ethyl cellulose, or cellulose acetate); malt: gelatin; oils (e.g., peanut oil, cottonseed oil. safflower oil, sesame oil, olive oil, com oil, or soybean oil); glycols; buffering agents; saline; Ringer's solution; alcohols; lubricants; coloring agents; dispersing agents; preservatives; or antioxidants.

[0109] “Pharmaceutically acceptable salt” refers to pharmaceutically acceptable organic or inorganic salts of a compound of an embodiment. Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate “mesylate”, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., l.l'-methylene-bis-(2-hydroxy-3- naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counter ion. The counter ion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counter ion.

[0110] “Polypeptide” and “protein” refer interchangeably to a compound of two or more subunit ammo acids, amino acid analogs, or other peptidomimetics, regardless of post- translational modification, e g., phosphorylation or glycosylation. The subunits may be linked by peptide bonds or other bonds such as, for example, ester or ether bonds. Full-length polypeptides, truncated polypeptides, point mutants, insertion mutants, splice variants, chimeric proteins, and fragments thereof are encompassed by this definition. In various embodiments thepolypeptides can have at least 10 amino acids or at least 25, or at least 50 or at least 75 or at least 100 or at least 125 or at least 150 or at least 175 or at least 200 amino acids.

[0111] '‘Progression-free survival’’ is well known to one of skill in the art and refers to the length of time during and after treatment in which a subject is living with a cancer that does not get worse, and can be used in a clinical study or trial to help find out how well a treatment is working.

[0112] “Reduced” and grammatical equivalents (including “lower.” “smaller.” etc.) when in reference to the expression of any characteristic in a first subject relative to a second subject, mean that the quantity and / or magnitude of the characteristic in the first subject is lower than in the second subject by any amount that is recognized as clinically relevant by any medically trained personnel. In one embodiment, the quantity and / or magnitude of the characteristic in the first subject is at least 10% lower than, at least 25% lower than, at least 50% lower than, at least 75% lower than, and / or at least 90% lower than the quantity and / or magnitude of the characteristic in the second subject. In embodiments, either the first or second subject may be treated and the other of the first or second subject may be untreated. In embodiments, the first subject is treated and the second subject is untreated.

[0113] '‘Small molecule” refers to a low molecular weight organic compound that may help regulate a biological process. Small molecules include any molecules with a molecular weight of about 2000 daltons or less, such as of about 500 to about 900 daltons or less. Small molecules can have a variety of biological functions, serving as cell signaling molecules, as drugs in medicine, and in many other roles. These compounds can be natural or artificial. Biopolymers such as nucleic acids and proteins, and polysaccharides (such as starch or cellulose) are not small molecules-though their constituent monomers, ribo- or deoxyribonucleotides, amino acids, and monosaccharides, respectively, are often considered small molecules. Small molecules include pharmaceutically acceptable salts of small molecules.

[0114] “Subject” refers to any mammal for whom diagnosis, treatment, or therapy is desired including mammals, e.g., humans, laboratory' animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), and horses.

[0115] '‘Substantially homogenous” refers to a population of cells derived from the same mammalian organ or region of a mammalian organ wherein the majority between about 100% to about 70%; betw een about 100% to about 90% of the total number of cells have a specified characteristic of interest.

[0116] “Therapeutically effective amount'’ is that amount sufficient, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease (e.g., cancer), condition, or disorder, and / or pharmacokinetic or pharmacodynamic effect of the treatment in a subject. A therapeutically effective amount can be administered in one or more administrations. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject. One skilled in the art will recognize that the condition of the individual can be monitored throughout the course of therapy and that the effective amount of a compound or composition disclosed herein that is administered can be adjusted accordingly.

[0117] “Trabecular number’" refers to the average number of trabeculae per unit length.

[0118] “Trabecular separation” refers to the mean distance between trabeculae.

[0119] “Treat,” “treating” or “treatment” refer to an action to obtain a beneficial or desired clinical result including, but not limited to, alleviation or amelioration of one or more signs or symptoms of a disease or condition (e.g., regression, partial or complete), diminishing the extent of disease, stability' (i.e.. not worsening, achieving stable disease) of the state of disease, amelioration or palliation of the disease state, diminishing rate of or time to progression, and remission (whether partial or total). “Treatment” of a cancer can also mean prolonging survival as compared to expected survival in the absence of treatment. Treatment need not be curative. In certain embodiments, treatment includes one or more of a decrease in pain or an increase in the quality of life (QOL) as judged by a qualified individual, e.g., a treating physician, e g., using accepted assessment tools of pain and QOL. In certain embodiments, a decrease in pain or an increase in the QOL as judged by a qualified individual, e.g., a treating physician, e.g., using accepted assessment tools of pain and QOL is not considered to be a “treatment” of the cancer. “Treat” covers any treatment of a cancer in a mammal, and includes: (a) preventing the cancer from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the cancer, i.e., arresting its development; or (c) relieving the cancer, i.e., causing regression of the cancer. The therapeutic composition may be administered before, during or after the onset of cancer. The therapy may be administered during the symptomatic stage of the cancer, and in some cases after the symptomatic stage of the cancer.

[0120] ‘Tumor microenvironment” or “cancer microenvironment” refers to the cellular environment or milieu in which the tumor or neoplasm exists, including surrounding blood vessels as well as non-cancerous cells including, but not limited to, immune cells, fibroblasts, bone marrow-derived inflammatory cells, and lymphocytes. Signaling molecules and theextracellular matrix also comprise the tumor microenvironment. The tumor and the surrounding microenvironment are closely related and interact constantly. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance, while the immune cells in the microenvironment can affect the growth and evolution of tumor cells.

[0121] In an aspect, the present disclosure provides an anticancer peptide comprise at least 90% sequence identity with an amino acid sequence selected from any of SEQ ID NO: 1- 10. In any embodiment, the amino acid sequence may be selected from SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5, or a sequence having at least 90% sequence identity thereto. In any embodiment, the amino acid sequence may comprise SEQ ID NO: 5, or a sequence having at least 90% sequence identity with SEQ ID NO: 5. In any embodiment, the sequence identity may be at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%. In any embodiment, the amino acid sequence may comprise SEQ ID NO: 4, or a sequence having at least 90% sequence identity with SEQ ID NO: 4. In any embodiment, the sequence identity may be at least 95%. In any embodiment, the sequence identity may be at least 99%. In any embodiment, the sequence identity may be 100%. In any embodiment, the sequence identity may be at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%.

[0122] In any embodiment of the anticancer peptide, one or more amino acids of the amino acid sequence may be modified. In any embodiment of the anticancer peptide, one or more amino acids of the amino acid sequence may be phosphorylated, glycosylated, nitrosylated, biotinylated, methylated, acetylated, or lipidated. In any embodiment wherein one or more amino acids of the amino acid sequence is glycosylated, the one or more glycosylated amino acids may be N-glycosylated, O-glycosylated, C-glycosylated, glypiated. or phosphoglycosylated.

[0123] In another aspect, the present disclosure provides a pharmaceutical composition comprising one or more anticancer peptides as described herein. In any embodiment, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, a pharmaceutically acceptable preservative, a pharmaceutically acceptable antioxidant, a pharmaceutically acceptable salt, a pharmaceutically acceptable pH modulator, a pharmaceutically acceptable buffer, or any combination thereof. In any embodiment, the anticancer peptide may comprise at least 99% purity. In any embodiment, the composition may be essentially free of peptides, nucleic acids, and cell fragments, other than the anticancer peptide.

[0124] In another aspect, the present disclosure provides a kit comprising the pharmaceutical composition as described herein; a container; a label; and instructions that provide methods of administering the pharmaceutical composition.

[0125] In still another aspect, the present disclosure provides a method of generating anticancer peptides, the method comprising: contacting mammalian cells with one or more cell growth signaling pathway activators; growing the mammalian cells in culture medium; removing the mammalian cells from the culture medium; adding a protein digestion enzyme to the culture medium; and purifying peptides of about 100 amino acids or less in length from the culture medium. The peptides purified from the medium may be 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20. 15, or 10 amino acids in length, or even less.

[0126] In any embodiment of the method, the mammalian cells may be mesenchymal stem cells (MSCs). In any embodiment, contacting mammalian cells with one or more cell growth signaling pathway activators may comprise contacting mammalian cells with two or more, three or more, four or more, or five or more cell growth signaling pathway activators. In any embodiment, the cell growth signaling pathway activators may be selected from Wnt signaling pathway activators, PI3K signaling pathway activators, FGF signaling pathway activators, and Notch signaling pathway activators. In any embodiment, the cell growth signaling pathway activators are selected from cMyc, Oct4, Klf4, and Sox2, or variants, homologs, derivatives, or fragments of any thereof. In any embodiment, the cell growth signaling pathway activators are selected from cMyc, Oct4, Klf4, and Sox2. In any embodiment, the protein digestion enzyme may be trypsin.

[0127] In any embodiment, removing the mammalian cells from the culture medium may comprise one or more rounds of centrifugation. In any embodiment, the centrifugation may be at from about 2,000 to about 4,000 rpm. The centrifugation may be at about 2,000 rpm. about 2,100 rpm, about 2,200 rpm, about 2,300 rpm, about 2,400 rpm, about 2,500 rpm, about 2,600 rpm, about 2,700 rpm, about 2,800 rpm, about 2,900 rpm, about 3,000 rpm, about 3,100 rpm, about 3,200 rpm, about 3,300 rpm, about 3,400 rpm, about 3,500 rpm, about 3,600 rpm, about 3,700 rpm, about 3,800 rpm. about 3,900 rpm, or about 4.000 rpm.

[0128] In any embodiment, the method may further comprise filtering the culture medium. In any embodiment, the filtering may be with a poly ethersulfone membrane. In any such embodiment, the poly ethersulfone membrane may be a 0.22-pm polyethersulfone membrane.

[0129] In any embodiment, the culture medium may comprise fetal bovine serum. In any embodiment, the culture medium may further comprise one or more antibiotics. In anyembodiment, the growing mammalian cells in culture medium may be performed at about 37°C. In any embodiment, the growing mammalian cells in culture medium may be performed at about 5% CO2 atmosphere.

[0130] In any embodiment, the purifying may comprise extracting via ion exchange chromatography, gel permeation chromatography, medium-pressure reversed phase chromatography, or high-pressure reversed phase chromatography.

[0131] In any embodiment, the method may further comprise eluting the purified peptides.

[0132] In still another aspect, the present disclosure provides a method of killing cancer cells, the method comprising: contacting the cancer cells with an anti cancer peptide as described herein. In any embodiment, the cancer cells may be human cancer cells. In any embodiment, the cancer cells may be selected from any of: osteosarcoma cells, breast cancer cells, or pancreatic cancer cells. In any embodiment, the cancer cells may be osteosarcoma cells. In any embodiment, the cancer cells may be breast cancer cells. In any embodiment, the cancer cells may be pancreatic cancer cells. In any embodiment, the cancer cells may be pancreatic ductal adenocarcinoma cells.

[0133] In another aspect, the present disclosure provides use of an anticancer peptide or pharmaceutical composition or composition as described herein for the manufacture of a medicament for the therapeutic treatment of cancer. In any embodiment of such use, the cancer may be osteosarcoma. In any embodiment of such use, the cancer may be breast cancer. In any embodiment of such use, the cancer may be pancreatic cancer. In any embodiment, the cancer may be pancreatic ductal adenocarcinoma.

[0134] In another aspect, the present disclosure provides a composition comprising a polynucleotide encoding an anticancer peptide as described herein. In any embodiment, the polynucleotide may be encoded in a vector. In any embodiment, the vector may be selected from the group consisting of plasmids, viral vectors, bacteriophages, cosmids, artificial chromosomes, and any combination thereof. In any embodiment, the viral vector may comprise an adeno-associated virus.

[0135] In another aspect, the present disclosure provides a kit comprising the composition comprising a polynucleotide encoding an anticancer peptide as described herein; a container; a label; and instructions that provide methods of administering the composition.

[0136] In yet another aspect, the present disclosure provides a method of treating a cancer in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of the anticancer peptide described herein or administering tothe subject a therapeutically effective amount of the pharmaceutical composition described herein or administering to the subject a therapeutically effective amount of the composition described herein.

[0137] In any embodiment of the method of treating a cancer in a subj ect in need thereof the subject may be a human. In any embodiment, the cancer may be selected from: osteosarcoma, breast cancer, or pancreatic cancer. In any embodiment, the cancer may be osteosarcoma. In any embodiment, the cancer may be breast cancer. In any embodiment, the cancer may be pancreatic cancer. In any embodiment, the cancer may be pancreatic ductal adenocarcinoma. In any embodiment, the cancer may be metastatic. In any embodiment, the cancer may be incipient. In any embodiment, treating the cancer may reduce a tumor size. In any embodiment, treating the cancer may increase bone volume ratio, increase bone mineral density, increase trabecular number, reduce trabecular separation, or reduce osteoclastogenesis, or a combination thereof. In any embodiment, treating the cancer may treat cancer-onset diabetes or cancer-related j aundice or both.

[0138] In another aspect, the present disclosure provides a kit comprising an anticancer peptide as described herein; a container; a label; and instructions that provide methods of administering the anticancer peptide.

[0139] Aspects of the present disclosure can be described as embodiments in any of the following enumerated clauses. It will be understood that any of the described embodiments can be used in connection with any other described embodiments to the extent that the embodiments do not contradict one another.CLAUSES

[0140] Clause 1. An anticancer peptide comprising at least 90% sequence identity with an amino acid sequence selected from any of SEQ ID NO: 1-23.

[0141] Clause 2. The anti cancer peptide of clause 1, wherein the amino acid sequence is selected from SEQ ID NO: 2, SEQ ID NO: 4. and SEQ ID NO: 5 or a sequence having at least 90% sequence identity thereto.

[0142] Clause 3. The anti cancer peptide of clause 1, wherein the amino acid sequence comprises SEQ ID NO: 5 or a sequence having at least 90% sequence identity with SEQ ID NO: 5.

[0143] Clause . The anti cancer peptide of clause 1, wherein the amino acid sequence comprises SEQ ID NO: 18 or SEQ ID NO: 23, or a sequence having at least 90%sequence identity with SEQ ID NO: 18, or a sequence having at least 90% sequence identity with SEQ ID NO: 23.

[0144] Clause 5. The anti cancer peptide of any of clauses 1-4, wherein the sequence identity' is at least 95%.

[0145] Clause 6. The anti cancer peptide of any of clauses 1-4, wherein the sequence identity’ is at least 99%.

[0146] Clause 7. The anticancer peptide of any of clauses 1-4. wherein the sequence identity' is 100%.

[0147] Clause 8. The anticancer peptide of any of clauses 1-7, wherein one or more amino acids of the amino acid sequence is phosphorylated, glycosylated, nitrosylated, biotinylated, methylated, acetylated, or lipidated.

[0148] Clause 9. The anticancer peptide of clause 8, wherein one or more amino acids of the amino acid sequence is glycosylated and the one or more glycosylated amino acids are N-glycosylated, O-glycosylated, C-glycosylated, glypiated, or phosphoglycosylated.

[0149] Clause 10. A pharmaceutical composition comprising one or more anticancer peptides of any of clauses 1-9 suspended in solution.

[0150] Clause 11. The pharmaceutical composition of clause 10, further comprising a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, pharmaceutically acceptable preservative, pharmaceutically acceptable antioxidant, pharmaceutically acceptable salt, pharmaceutically acceptable pH modulator, pharmaceutically acceptable buffer, or any combination thereof.

[0151] Clause 12. The pharmaceutical composition of either of clauses 10 and 11, wherein the anticancer peptide comprises at least 99% purity.

[0152] Clause 13. The pharmaceutical composition of either of clauses 10 and 11, w herein the composition is essentially free of peptides, nucleic acids, and cell fragments, other than the anticancer peptide.

[0153] Clause 14. A kit comprising: the pharmaceutical composition of any of clauses 10-13; a container; a label; and instructions that provide methods of administering the pharmaceutical composition.

[0154] Clause 15. A method of generating anticancer peptides, the method comprising: contacting mammalian cells with one or more cell growth signaling pathway activators; growing the mammalian cells in culture medium; removing the mammalian cells from the culture medium; adding a protein digestion enzyme to the culture medium; and purifying peptides of about 100 amino acids or less in length from the culture medium.

[0155] Clause 16. The method of clause 15, wherein the mammalian cells are mesenchymal stem cells (MSCs).

[0156] 17. The method of clause 15 or clause 16, wherein contacting mammalian cells with one or more cell growth signaling pathway activators comprises contacting mammalian cells with two or more, three or more, four or more, or five or more cell growth signaling pathway activators.

[0157] Clause 18. The method of any of clauses 15-17. wherein the cell growth signaling pathway activators are selected from Wnt signaling pathway activators, PI3K signaling pathw ay activators, FGF signaling pathway activators, and Notch signaling pathway activators.

[0158] Clause 19. The method of any of clauses 15-18. wherein the cell growth signaling pathway activators are selected from cMyc, Oct4, Klf4, and Sox2, or variants, homologs, derivatives, or fragments of any thereof.

[0159] Clause 20. The method of any of clauses 15-19, wherein the cell growth signaling pathway activators are selected from cMyc, Oct4, Klf4, and Sox2.

[0160] Clause 21. The method of any of clauses 15-20, wherein the protein digestion enzyme is trypsin.

[0161] Clause 22. The method of any of clauses 15-21, wherein removing the mammalian cells from the culture medium comprises one or more rounds of centrifugation.

[0162] Clause 23. The method of any of clauses 15-22. further comprising filtering the culture medium.

[0163] Clause 24. The method of any of clauses 15-23, wherein the culture medium comprises fetal bovine serum.

[0164] Clause 25. The method of any of clauses 15-24, wherein the culture medium further comprises one or more antibiotics.

[0165] Clause 26. The method of any of clauses 15-25, wherein the growing mammalian cells in culture medium is performed at about 37°C.

[0166] Clause 27. The method of any of clauses 15-26. wherein the growing mammalian cells in culture medium is performed at about 5% CO2 atmosphere.

[0167] Clause 28. The method of any of clauses 23-27, wherein the filtering is with a 0.22-pm polyethersulfone membrane.

[0168] Clause 29. The method of any of clauses 22-28, wherein the centrifugation is at from about 2,000 to about 4,000 rpm.

[0169] Clause 30. The method of any of clauses 15-29, wherein the purifying comprises extracting via ion exchange chromatography, gel permeation chromatography, medium-pressure reversed phase chromatography, or high-pressure reversed phase chromatography.

[0170] Clause 31. The method of any of clauses 15-30, further comprising eluting the purified peptides.

[0171] Clause 32. A method of treating a cancer in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of the anticancer peptide of any of clauses 1-9; or administering to the subject a therapeutically effective amount of the pharmaceutical composition of any of clauses 10-13; or administering to the subject a therapeutically effective amount of the composition of any of clauses 46-49.

[0172] Clause 33. The method of clause 32, wherein the subject is a human.

[0173] Clause 34. The method of clause 32 or clause 33, wherein the cancer is osteosarcoma.

[0174] Clause 35. The method of clause 32 or clause 33, wherein the cancer is breast cancer.

[0175] Clause 36. The method of any of clauses 32-35, wherein the cancer is metastatic.

[0176] Clause 37. The method of any of clauses 32-36, wherein treating the cancer reduces a tumor size.

[0177] Clause 38. The method of any of clauses 34-37, wherein treating the cancer increases bone volume ratio, increases bone mineral density, increases trabecular number, reduces trabecular separation, or reduces osteoclastogenesis, or a combination thereof.

[0178] Clause 39. A method of killing cancer cells, the method comprising: contacting the cancer cells with the anticancer peptide of any of clauses 1-9.

[0179] Clause 40. The method of clause 39, wherein the cancer cells are human cancer cells.

[0180] Clause 41. The method of clause 39 or clause 40. wherein the cancer cells are osteosarcoma cells.

[0181] Clause 42. The method of clause 39 or clause 40, wherein the cancer cells are breast cancer cells.

[0182] Clause 43. Use of an anticancer peptide of any of clauses 1-9; or a pharmaceutical composition of any of clauses 10-13; or a composition of any of clauses 46-49 for the manufacture of a medicament for the therapeutic treatment of cancer.

[0183] Clause 44. The use of clause 43, wherein the cancer is osteosarcoma.

[0184] Clause 45. The use of clause 43, wherein the cancer is breast cancer.

[0185] Clause 46. A composition comprising a polynucleotide encoding an anticancer peptide of any of clauses 1-7.

[0186] Clause 47. The composition of clause 46, wherein the polynucleotide is encoded in a vector.

[0187] Clause 48. The composition of clause 47. wherein the vector is selected from the group consisting of plasmids, viral vectors, bacteriophages, cosmids, artificial chromosomes, and any combination thereof.

[0188] Clause 49. The composition of clause 48, wherein the viral vector comprises an adeno-associated virus.

[0189] Clause 50. A kit comprising: the composition of any of clauses 46-49; a container; a label; and instructions that provide methods of administering the composition.

[0190] Clause 51. The method of clause 32 or clause 33, wherein the cancer is any of osteosarcoma, breast cancer, or pancreatic cancer.

[0191] Clause 52. The method of clause 32 or clause 33, wherein the cancer is pancreatic cancer.

[0192] Clause 53. The method of clause 52, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

[0193] Clause 54. The method of any of clauses 32, 33, or 51-53, wherein the cancer is incipient.

[0194] Clause 55. The method of any of clauses 51-54, wherein treating pancreatic cancer treats cancer-onset diabetes or cancer-related jaundice or both.

[0195] Clause 56. The method of clause 39 or clause 40. wherein the cancer cells are pancreatic cancer cells.

[0196] Clause 57. The method of clause 55 wherein the pancreatic cancer cells are pancreatic ductal adenocarcinoma cells.

[0197] Clause 58. The use of clause 43 wherein the cancer is pancreatic cancer.

[0198] Clause 59. The use of clause 58 wherein the cancer is pancreatic ductal adenocarcinoma.

[0199] Clause 60. A kit comprising: the anticancer peptide of any of clauses 1-9; a container; a label; and instructions that provide methods of administering the anticancer peptide.

[0200] Clause 61. The anticancer peptide of clause 1, wherein the amino acid sequence comprises SEQ ID NO: 4 or a sequence having at least 90% sequence identity with SEQ ID NO: 4.

[0201] Clause 62. The anti cancer peptide of clause 61, wherein the sequence identity is at least 95%.

[0202] Clause 63. The anti cancer peptide of clause 61, wherein the sequence identity is at least 99%.

[0203] Clause 64. The anti cancer peptide of clause 61, wherein the sequence identity is 100%.

[0204] Clause 65. An anticancer peptide, a pharmaceutical composition, a kit, a method, or a use as substantially described herein.

[0205] Clause 66. An anticancer peptide comprising at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 18 or SEQ ID NO: 23.

[0206] Clause 67. An anti cancer pharmaceutical composition comprising the anticancer peptide of clause 66.

[0207] Clause 68. The anticancer peptide of clause 66. wherein the peptide comprises an N-terminus acetyl modification, a C-terminus amino-group modification, or both an N-terminus acetyl modification and a C-terminus amino-group modification.

[0208] Clause 69. The anticancer pharmaceutical composition of clause 67, further comprising cisplatin, taxol, or both cisplatin and taxol.

[0209] Clause 70. An anticancer pharmaceutical composition comprising the anticancer peptide of clause 68.

[0210] Clause 71. The anticancer pharmaceutical composition of clause 70, further comprising cisplatin, taxol, or both cisplatin and taxol.EXAMPLES

[0211] Examples related to the present disclosure are described below. In some embodiments, alternative techniques can be used. The example is intended to be illustrative and are not limiting or restrictive of the scope of the claimed subject matter. Other variations or embodiments will be apparent to a person of ordinary skill in the art from the above-description.Example 1. Pancreatic Cancer.

[0212] Pancreatic ductal carcinoma (PDAC) is a highly aggressive malignant tumor. Induced tumor-suppressing cells (iTSCs) were previously developed that secret a group of tumor-suppressing proteins. Here, we examined a unique procedure to identify anticancer peptides (ACPs), using trypsin-digested iTSCs-derived protein fragments. Among the ten ACP candidates (designated P01, P02, P03, P04, P05, P06, P07, P08, P09, and PIO), P04 (IGEHTPSSLAIMENANVLAR) presented the most efficient anti-PDAC activities. P04 was derived from Aldolase A (ALDOA), a glycolytic enzyme. Extracellular ALDO A, as well as P04, was predicted to interact with epidermal growth factor receptor (EGFR), and P04 downregulated oncoproteins such as Snail and Src. Importantly, P04 has no inhibitor)' effect on mesenchymal stem cells (MSCs).

[0213] We also generated iTSCs by overexpressing ALDOA in MSCs and peripheral blood mononuclear cells (PBMCs). iTSC-derived conditioned medium (CM) inhibited the progression of PDAC cells as well as PDAC tissue fragments. The inhibitor}7effect of P04 was combinatorial to that of CM and chemotherapeutic drugs such as 5 -Flu and Gemcitabine. Notably, applying mechanical vibration to PBMCs elevated ALDOA and phosphorylated Akt in PI3K signaling and converted PBMCs into iTSCs. Collectively, this study presented a unique procedure for selecting anticancer P04 from ALDOA in an iTSCs-derived proteome for the treatment of PDAC.

[0214] Introduction.

[0215] Pancreatic ductal adenocarcinoma (PDAC) stands as the most prevalent and aggressive form of pancreatic cancer, constituting approximately 85 to 90 percent of all instances. The five-year survival rate from time of diagnosis of PDAC is about 6 percent, and median survival from time of diagnosis is only about six months. Despite relatively low- incidence, PDAC is the third most common cause of cancer-related death in the United States. The high mortality rate for PDAC is attributed to lack of early diagnosis and ineffective treatment options for advanced tumors. Under the current standard of care for treating PDAC, remission and cure is not considered possible, and treatment is only expected to add months to a patient's anticipated survival.

[0216] Nestled deep within the abdominal cavity, the pancreas poses a challenge for early PDAC detection. Initial stages of PDAC often remain inconspicuous, and when symptoms do manifest, they frequently signal an advanced tumor beyond the point of surgical removal. Up to 80 percent of PDAC patients present with advanced and / or metastatic disease at initial diagnosis. Surgical resection offers the greatest efficacy for those with early-diagnosed PDAC. To address PDAC at this stage, FOLFIRINOX (also sometimes called FOLFOXIRI), anadjuvant chemotherapy regimen, incorporating fluorouracil (5-Flu). leucovorin, irinotecan, and oxaliplatin, is the recommended course of action. Nonetheless, PDAC frequently develops resistance to chemotherapy. While radiation treatment can mitigate pain, its ability to impede PDAC progression often proves ineffective. For patients whose PDAC presents as non- resectable, the best available treatment is chemotherapy and radiation followed by revaluation in the hope that tumor size is reduced enough that surgery becomes feasible.

[0217] Due to their distinct manufacturing process and superior quality control when com-pared to other cell and protein-based agents, anticancer peptides (ACPs) are emerging as highly promising candidates for cancer treatment, including PDAC treatment. These peptides typically comprise basic and hydrophobic residues, earn ing positive charges, which aligns with the negatively charged nature of most cancer cells. Despite the availability of various databases such as CancerPPD, ImmunoPeptidome, and PeptideCutter, which aid in the design of ACP candidates and the prediction of their tumor-binding epitopes, the effective design of ACPs remains a formidable challenge. Specifically, identifying precise targets and discerning cancer cells from normal cells continue to pose significant obstacles.

[0218] For the treatment of PDAC, ten ACP candidates were selected and tested. These ACPs were derived from tumor-suppressing proteins secreted from induced tumor-suppressing cells (iTSCs). It was previously shown that iTSCs and their tumor-suppressive conditioned medium (CM) can be generated from cancer and non-cancer cells by modulating varying signaling pathways. Interestingly. iTSCs can be generated by the overexpression of cMyc or Oct4, whereas the generation of induced pluripotent stem cells (iPSCs) requires the overexpression of four transcription factors, such as cMyc, Oct4, Klf4, and Sox2. Previous findings also demonstrated that CM of mesenchymal stem cells (MSCs) overexpressing Akt, and (3-catenin selectively inhibited tumor cell proliferation and invasion. (See Sun, X. et al. Preventing tumor progression to the bone by induced tumor-suppressing MSCs. Theranostics 2021, 11, 5143-5159.) Furthermore, the activation of PKA signaling in Jurkat T lymphocytes or inhibition of AMPK signaling in human peripheral blood mononuclear cells (PBMCs), were shown effective in generating iTSCs. (Jurkat cells, sometimes calls JM cells, are an immortalized T lymphocyte cell line used in cell culture.) Notably, the proteomic analysis showed that CM was enriched with proteins such as enolase 1, ubiquitin C, Calreticulin, and aldolase A (ALDOA), which may act as both pro and anticancer proteins depending on their intra-cellular and extracellular locations.

[0219] In this Example, the ten prospective ACP candidates (P01 to PIO) were selected from trypsin-digested peptides that were derived from iTSCs-derived CMs. In a cell-viabilityprescreening process, P04 showed promising anticancer capabilities and thus this Example focused on P04.

[0220] P04 was derived from ALDOA, which is an enzyme involved in a glycolytic pathway. ALDOA is highly expressed in cancer tissues, and its elevated expression level usually indicates a poor prognosis. Paradoxically, however, extracellular ALDOA acts as a tumorsuppressing protein in breast cancer. We investigated in this study whether ALDOA-derived P04 is a potential candidate for the treatment of PDAC, and whether it may act combinatorially with chemotherapeutic agents such as Fluorouracil (5-Flu) and Gemcitabine (Gem).

[0221] In addition to the utilization of P04, our approach encompassed the generation of induced tumor-suppressing cells (iTSCs) and the production of tumor-suppressive conditioned medium (CM) via two distinct methodologies: ALDOA overexpression and the application of low-intensity vibrations (LIV). Similar to Akt within the PI3K signaling cascade and P-catenin within the Wnt signaling pathway, we conjectured that the activation of oncogenic ALDOA within the intracellular domain could potentially yield iTSCs and engender the creation of tumor-suppressing CM. Furthermore, we postulated that mechanical stimuli, like LIV. could potentially prompt the generation of iTSCs by potentially inciting the activation of oncogenic PI3K signaling.

[0222] Materials and Methods.

[0223] Cell Culture

[0224] Human pancreatic tumor cell lines such as PANCI (ATCC, Manassas, VA, USA), PANC198 and Pa03C (obtained from Indiana University Simon Comprehensive Cancer Center, Indianapolis, USA) were cultured in DMEM (10-013-CV, Coming Inc. Coming, NY, USA). MDA-MB-231 and MDA-MB-436 breast cancer cell lines (ATCC) were grown in RPMI-1640 (Gibco, Carlsbad, CA, USA). The culture medium was supplemented with 10% fetal bovine serum and antibiotics (100 units / mL penicillin, and 100 pg / mL streptomycin; Life Technologies, Grand Island, NY, USA). Jurkat T lymphocytes (ATCC) were cultured in RPMI1640 (10-040-CV, Coming). Murine mesenchymal stem cells (MSCs) derived from the bone marrow of the C57BL / 6 mice were cultured in DMEM. Cells were maintained at 37°C and 5% CO2. The overexpression of ALDOA was conducted by transfecting its plasmids (109854; Addgene, Cambridge, MA, USA), while blank plasmids (FLAG-HA-pcDNA3.1; Addgene) were used as a control.

[0225] PDAC cells were treated with 10 candidate peptides (P01 to P10, Genscript Biotech, Piscataway. NJ. USA) and chemotherapeutic agents, such as gemcitabine (3259, Tocris Bioscience, Minneapolis, MN, USA) and 5 -fluorouracil (3257, Tocris Bioscience). PDAC cellswere also treated with human recombinant ALDOA protein (MBS 8248528, MyBioSource, San Diego, CA, USA). For in vitro experiments, CM was prepared using low-speed centrifugation at 2,000 rpm for 10 minutes. The supernatants were further centrifuged at 4,000 rpm for 10 minutes and subjected to filtration with a 0.22-pm polyethersulfone membrane (Sigma, St. Louis, MO, USA).

[0226] MTT Assay and Ed U Assay

[0227] A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT)-based metabolic activity assay was evaluated using -2,000 cells seeded in 96-well plates. Cells were incubated for two days with treatment agents. They were dyed with 0.5 mg / mL thiazolyl blue tetrazolium bromide (M5655, Sigma) on day 4. Optical density' for assessing metabolic activities was determined at 562 nm. A 5-ethynyl-2'-deoxyuridine (EdU)-based proliferation activity assay was evaluated using -1,000 cells, which were seeded in 96-well plates. We employed a fluorescence-based cell proliferation kit (Click-iT™, EdU Alexa Fluor™ 488 Imaging Kit; Thermo Fisher, Waltham, MA, USA), using the procedure described by the manufacturer.

[0228] Scratch Assay

[0229] A wound-healing scratch assay was utilized to evaluate the two-dimensional migratory behavior of PANC cells by determining the change in the scratch area. Approximately 3x l03cells were seeded in 12-well plates on day 1. On day 2, a scratch was made on the cell layer with a tip of a plastic pipette. Cells were washed with Dulbecco's Modified Eagle Medium (DMEM) to remove floating cells. Images of cell-free zones were taken at 0 hours and 24 hours with a light microscope (40 x), and the areas of the cell-free zone were quantified by ImageJ software.

[0230] Transwell® Invasion Assay

[0231] The invasion capacity of tumor cells was determined using a 12-well plate and Transwell® chambers (353182, Thermo Fisher) with 8-pm pore size. The chambers were coated with 300 pL Matrigel® (100 pg / mL), and 500 pL of the serum-free medium was added. The chamber was washed three times with the serum-free medium. Approximately 7xl04cells in 300 pL serum-free DMEM were placed in the upper chamber, and 800 pL CM was added to the lower chamber. Cells, which invaded the lower side of the membrane, were fixed, and stained with methanol and crystal violet. Five randomly chosen images were taken with an inverted optical microscope (100x), and the average number of stained cells, which represented the invasion capacity, was determined.

[0232] Western Blot Analysis

[0233] Cells were lysed with a radioimmunoprecipitation assay (RIP A) lysis buffer (sc-24948, Santa Cruz Biotech, Dallas, TX, USA) with protease inhibitors (PIA32963.Thermo Fisher), and phosphatase inhibitors (2006643, Calbiochem™, Billerica, MA, USA). Proteins were separated by 10%— 15% sodium dodecyl sulfate (SDS) gel (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to a poly vinylidene difluoride (PVDF) membrane (IPVH00010. Millipore, Billerica, MA, USA). The membrane was incubated with the primary antibodies, followed by incubation with secondary antibodies (7074S / 7076S, Cell Signaling, Danvers, MA, USA). We used antibodies against ALDOA, Snail, p-Src, Src, cleaved caspase-3, caspase-3 (Cell Signaling), and K-Ras (Santa Cruz Biotech) with p-actin (Sigma) as a control. The protein level was determined using a SuperSignal west femto maximum sensitivity substrate (PI34096, Thermo Fisher), and a luminescent image analyzer (LAS-3000, Fuji Film, Tokyo, Japan) was used to quantify signal intensities.

[0234] Human Peripheral Blood Mononuclear Cells (PBMCs)

[0235] Human peripheral blood samples were used based on the guidelines of the Declaration of Helsinki. The use of blood samples was approved by the Ethics Committee of Osaka University (protocol #21344). Eight mL of peripheral blood was collected from 10 healthy volunteers (mean of 34.2 years, ranging from 23 to 54 years). An equal volume of 0.9% NaCl solution was mixed with blood samples, and 6 mL of the mixed solution was added with 3 mL of a lymph prep solution (1114544, Abbott Diagnostics Technologies AS, Norway). The mix solution was centrifuged at 800xg for 30 minutes at room temperature. Using a Pasteur pipette, PBMCs were carefully collected, and they were cultured in AlyS705 medium (Cell Science & Technology Institute, Inc., Sendai, Japan).

[0236] Ex-Vivo Tissue Assay

[0237] The usage of human pancreatic cancer was approved by the Indiana University Institutional Review Board (1911155674), and the tissues were received from Indiana University Simon Comprehensive Cancer Center Tissue Procurement Core. A freshly isolated pancreatic cancer tissue (~ 1 gram) was manually fragmented with a scalpel into small pieces (0.5 ~ 0.8 mm in length), which were grown in DMEM with 10% fetal bovine serum and antibiotics for one day. The described CM was then added for 4 additional days, and a change in the fragment size was determined.

[0238] Molecular Docking Analysis

[0239] To evaluate interactions between epidermal growth factor receptor (EGFR) and ALDOA, as well as EGFR and P05, a ZDOCK program (ver. 2016, Discovery Studio, SanDiego, CA, USA) was employed. All possible binding poses were considered in the translational and rotational space between EGFR and ALDOA or P05, and each pose was evaluated by determining the energy-based ZDOCK scoring system. Hydrogen-bonding interactions were predicted for the EGFR-ALDOA complex and EGFR-P05 complex.

[0240] Vibration Assay

[0241] To generate an anti-PDAC conditioned medium (CM), vertical low-intensity vibration (LIV. 90 Hz at 2 g-level) was applied to Jurket cells and PBMCs (~3x 105cells) for 20 minutes twice with a 3-hour interval. After the second LIV, the culture medium was exchanged for the fresh medium. Cells were incubated for 24 hours and CM was harvested. To elevate the viscosity of the culture medium from 0.8 cP to 20 cP, 0.1% of methylcellulose solution (#428430500, Thermo Fisher) was added to the medium.

[0242] Statistical Analyses

[0243] For cell-based experiments, three or four independent experiments were conducted, and data were expressed as mean ± S.D. Statistical significance was evaluated using a one-way analysis of variance (ANOVA). Post-hoc statistical comparisons with control groups were performed using Bonferroni correction with statistical significance at p < 0.05. The single and double asterisks in the figures indicate p < 0.05 and p < 0.01, respectively.

[0244] Results.

[0245] Anti-Tumor Effect ofP02, P04, and P07 In Pancreatic Ductal Adenocarcinoma Cells

[0246] We first evaluated the ten potential anti-tumor peptides (designated as P01 to P10), which were selected from the conditioned medium (CM) derived from MSCs. Based on our previous studies, these MSCs were engineered to express anti-tumor proteins (Table 1).

[0247] The selected peptides were obtained from trypsin-digested fragments of all proteins present in the CM, and their ability to inhibit cancer grow th was evaluated using three PDAC cell lines (PANCI, PAN198, and PA03C). Out of all the peptides tested, the peptides, P02, P04, and P07. exhibited a high reduction in cell viability on average (Table 2).

[0248] The amino acid sequences of these three peptides were listed (Fig. 1A). Of the three peptides examined, P04 demonstrated the strongest anti-tumor effects, particularly in terms of reducing MTT-based cell viability and inhibiting scratch-based cell motility of PANCIcells (Fig. IB). Subsequently, we examined the inhibitory effects of these three peptides on the viability and motility. Our scratch assay results revealed that P02, P04. and P07 reduced the motility of PANCI cells, and P04 exhibited the strongest anti-motility effect (Fig. 1C). Hereafter, we focused on P04 and tested its impact on the proliferation and invasion of pancreatic cancer cells. The EdU assay and Transwell® invasion assay showed that P04 decreased the proliferation and invasive properties of PANCI cells (Fig. 1D&E).

[0249] Anti-Tumor Effect ofP04 On Human PDAC Tissue Fragments

[0250] To further investigate the efficacy of P04 as an anti -tumor agent, we exposed fresh human pancreatic cancer tissue fragments to P04. As shown in the illustration (Fig. 2A), we collected PDAC tissue from a patient and divided it into small fragments. One group was cultured in a standard medium, while the other was treated with 25 pg / ml of P04. We cultured both groups for four days and monitored their shapes every 48 hours (Fig. 2A). The results revealed that P04 reduced the size of ex vivo PDAC fragments for 4 days (Fig. 2B&C). We also observed that the combination of P04 and 5 -Flu was more effective than 5-Flu alone in decreasing the size of PDAC fragments (Fig. 2D&E).

[0251] Enhanced Anti-Tumor Effects In Combination with Chemotherapeutic Agents

[0252] We further investigated the combined effects of P04 with tw o commonly used chemotherapeutic agents, Gemcitabine, and 5-Flu. The addition of P04 at a concentration of 25 pg / ml resulted in a decrease in the ICso value of Gemcitabine from 1.0 pM to 0.8 pM and of 5- Flu from 3.3 pM to 1.6 pM in the MTT-based viability assay of PANCI cells (Fig. 3A). Furthermore, the administration of P04 resulted in the downregulation of oncogenic proteins including p-Src and Snail in PANCI and PAN198 cells. Furthermore, it presented cytotoxicity by increasing the level of cleaved caspase-3, a marker of apoptosis (Fig. 3B).

[0253] Generation of Anti-Tumor Conditioned Medium By The Overexpression of ALDOA In Mesenchymal Stem Cells

[0254] ALDOA is a cytoplasmic enzyme that catalyzes the reversible conversion of fructose- 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate during glycolysis. The TCGA database showed that elevated transcript level of Aldoa in PDAC tissues was associated with a poor survival rate (Figs. 4A&B). Our previous studies indicated that many tumor-suppressing proteins, which were enriched in tumor-suppressing CM such as Moesin and Enolase 1, can covert tumor and non-tumor cells into iTSCs that generate tumorsuppressing CM. Here, ALDOA w as overexpressed in MSCs and the antitumor ability of their CM was examined. Interestingly, ALDOA-overexpressing MSC-derived CM presented tumor-suppressing capabilities, by reducing viability, proliferation, motility, and invasion of PANCI cells (Figs. 4C-E), as well as PANCI 98 cells (Figs. 9A-C).

[0255] The application of recombinant human ALDOA protein (rhALDOA) reduced MTT-based cell viability (Fig. 5A). Also, the Western blotting result showed that the levels of ALDOA and K-Ras were decreased in response to the application of rhALDOA (Fig. 5B). We have previously shown that the antitumor capability’ of ALDOA is mediated by its interaction with EGFR. A protein docking analysis in this study predicted that P04. a peptide fragment derived from ALDOA, may bind with EGFR with 4 putative hydrogen bondings at 2.68 A to 3.07 A (Fig. 5C, Table 3).

[0256] Generation of Tumor-Suppressive CM From Pbmcs by Overexpression of ALDOA

[0257] We next tested whether anti-PDAC CM can be generated from patient-derived PBMCs by the overexpression of ALDOA. ALDOA was found to increase after ALDOA plasmid transfection (Fig. 6A). Our results revealed that PBMC-derived CM decreased the MTT-based viability’ of PANCI and PAN198 cells (Fig. 6B). Furthermore, the anti-tumor effect of PBMC-derived CM was confirmed using PDAC tissue fragments. The inhibitory' effect of PBMC-derived CM was combinatorial to that of P04 (Fig. 6C&D).

[0258] Generation of Tumor-Suppressive CM Using Low -Intensity Vibration

[0259] We lastly examined whether anti-PANCl CM can be derived from PBMCs by the application of LIV (90 Hz with a g-level of 2). The result showed that LIV -treated lymphocyte-derived CM decreased the MTT-based viability of PANCI cells (Fig. 7A). The Western blot analysis revealed that LIV elevated the level of ALDOA in PBMCs (Fig. 7B), and their LIV -treated CM reduced MTT-based viability and scratch-based motility of PANCI cells (Fig. 7C). Importantly, the PBMC-derived CM did not reduce the viability and motility' of MSCs cells (Fig. 7D). Furthermore, applying mechanical vibration to Jurkat cells elevated phosphorylated Akt expression (Fig. 10A). The antitumor capability of LIV-treated CM wasmodulated by viscosity of the culture medium. In response to the increase in viscosity, the result showed the enhancement of the inhibition of the MTT-based viability’ of PANCI and PAN198 cells in response to LIV-treated lymphocyte-derived CM (Fig. 8A&B), as well as MDA-MA- 231 and MDA-MB-436 cells (Fig. 10B). Consistently, the same enhancement was observed in the inhibition of the scratch-based motility of PANCI cells (Fig. 8C).

[0260] Discussion.

[0261] In this Example, we showed that ALDOA-derived ACP P04 inhibited the viability, proliferation, migration, and invasion of human PANCI and PAN198 cells, and reduced the volume of patient-derived pancreatic cancer tissue fragments. The combined effect of P04 and a chemotherapeutic drug such as 5-Flu and Gemcitabine was combinatorial, suggesting a possible reduction in the current drug dosage. The administration of P04 downregulated p-Src and snail, while it upregulated the cytotoxic form of caspase-3. Importantly, intracellular ALDOA was highly expressed in PDAC cells and oncogenic, whereas the application of rhALDOA protein in the extracellular domain acted as an inhibitor of PDAC cells. The overexpression of ALDOA in MSCs and PMBCs converted these cells into iTSCs, whose CM significantly inhibited the viability of PDAC cells. Furthermore, the application of LIV was effective in generating iTSCs. LIV upregulated the expression of ALDOA in PBMCs, and their CM acted as a tumor-suppressing agent. The elevated viscosity of the culture medium enhanced the anti-tumor capability of LIV-treated PBMC-derived CM.

[0262] We used a protein docking program Z-DOCK to predict the possibility of P04 binding to EGFR. There were 4 possible interactions, in the range of 2.6 A to 3.0 A, between P04 and EGFR. Of note, we previously predicted the potential binding of EGFR to P05, which is the other ACP derived from ALDOA. Cui C et al. Anticancer peptides from induced tumorsuppressing cells for inhibiting osteosarcoma cells. Am. J. Cancer Res. 2023 (in press). A downstream signaling pathway of EGFR includes K-Ras, which is a small GTPase in the receptor tyrosine kinase family. The result showed that the application of P04 decreased K-Ras expression in PANCI cells. The abnormal activation of K-Ras is common in PDAC patients, which has a significant impact on clinical prognosis. Taken together, this study indicated that the anti-PDAC action of P04 is mediated by EGFR. followed by the inhibition of K-Ras signaling.

[0263] Compared to other ACP studies, ACP candidates in this study are unique since they were derived from iTSC-secreted tumor-suppressing proteins. For example, to prevent tumor-linked angiogenesis, VEGFR1 epitope peptide (SYGVLLWEI) (SEQ ID NO.: 24) and VEGFR2 epitope peptide (RFVPDGNRI) (SEQ ID NO.: 25) were developed. However, anti-VEGF therapy alone cannot usually cure PDAC. P04 can be used in combination with chemotherapeutic agents. The isoelectric point of P04 is 5.3, in comparison to 10.95 for VEGF1 / 2 peptides. P04 is closer to the isoelectric point of pancreatic tissue that is slightly acidic, suggesting superior solubility, stability, and biological activity'.

[0264] The generation of iTSCs by overexpressing ALDOA is paradoxical since a glycolytic enzyme, ALDOA, is highly expressed in various tumor types and associated with tumorigenesis and progression. However, the counterintuitive approach of inducing tumorsuppressive CM is a unique feature of iTSCs, and we have shown that the overexpression of oncogenes such as cMyc and K-Ras converts non-tumor cells such as MSCs and PBMCs into iTSCs. Consistently, intracellular ALDOA acts as oncogenic, while extracellular ALDOA behaves as anti-oncogenic. Compared to cell-based therapies, CM derived from MSCs and PBMCs is anticipated to present low immunogenicity, although it is preferable to use autologous MSCs and PBMCs.

[0265] Applying LIV converted PBMCs into iTSCs, supporting importance of the external mechanical environment on tumor suppression. Recently, LIV is reported to inhibit the proliferation of tumor cells by interfering with cell cycle signaling. Furthermore. LIV is known to promote the activity of immune cells. In this study, we generated iTSCs from PBMCs harvested from healthy individuals as well as patients with PDAC. The use of patient-derived PBMCs should lower the risk of immune rejection. Interestingly, increasing viscosity of culture medium enhanced the anti -tumor effect of LIV-treated iTSC CM. The high viscosity may elevate sensitivity of PBMCs to LIV by altering mechanical properties of PBMCs, such as membrane rigidity, and stiffness of cytoskeleton. Viscosity may also affect cell morphology' and cell-matrix interactions, as w ell as synthesis and secretion of proteins. Overall. LIV is a useful procedure to generate iTSCs without using any chemical or biological agents.

[0266] PBMCs can be obtained from the peripheral blood of patients with PDAC without any surgery. In this study, we converted PBMCs from PDAC patients into iTSCs by overexpressing ALDOA or applying LIV. The antitumor capability of iTSC CM was shown suing PDAC tissue fragments from the same patient. Furthermore, the combination of P04 with chemotherapeutic agents and iTSC CM increased the anti-tumor effect and decreased ICso of chemotherapeutic agents. The combinatorial strategy7may inhibit tumor progression through multiple signaling pathways by targeting different molecules, leading to a robust therapeutic effect. Collectively , the use of patient-derived PBMCs for developing therapeutic agents may present potential advantages. In contrast to alternative biological treatments, ACPs offer notable advantages, encompassing synthetic cost-effectiveness and stringent quality control measures.

[0267] Conclusions.

[0268] This Example establishes candidate anticancer peptides such as P04 as a potent anti-PANC ACP, effectively curbing the viability, motility, and invasive potential of three distinct PDAC cell lines. P04 seamlessly integrates with prevailing chemotherapeutic agents, substantially diminishing the IC50 values associated with each of the three agents under scrutiny. The anticancer efficacy of P04 is rooted in targeted mechanisms and not reliant on indiscriminate electrostatic interactions with negatively charged cancer cells.Example 2. Osteosarcoma and Breast Cancer.

[0269] For the treatment of osteosarcoma (OS), ACP candidates were selected and their tumor-suppressive capabilities were examined. Ten ACP candidates were derived from tumorsuppressing proteins that were secreted from a unique type of cells named induced tumorsuppressing cells (iTSCs). We have shown that the activation of cMyc paradoxically converts cancer cells as well as non-cancer cells into iTSCs, and iTSC-derived conditioned medium (CM) is enriched with tumor-suppressing proteins such as Calreticulin, Enolase 1, and Moesin. The question we addressed here was whether effective ACPs can be generated from proteins that were enriched in iTSC-derived CM. To the best of our knowledge, little is known about ACPs that are effective for the treatment of OS.

[0270] Introduction.

[0271] The generation of iTSCs by activating oncogenic signaling, such as cMyc and b- catenin / Wnt signaling, is linked to an unsuccessful reprogramming of induced pluripotent stem cells (iPSCs). To produce iPSCs, the transfection of the four transcription factors such as cMyc, Oct4, Klf4, and Sox2 is necessary. Importantly, however, the successful reprogramming rate of adult somatic cells is only about 1%, and a majority of transfected cells fail to acquire pluripotency. One type of such unsuccessfully transfected cells becomes oncogene-induced senescent (OIS) cells that secrete tumor-suppressive proteins. While it is unclear whether all iTSCs are senescent, mounting evidence suggests that the overexpression of oncoproteins and the activation of oncogenic pathways facilitate the secretion of anti -cancer proteins.

[0272] In this Example, we created try psin-digested peptides using iTSC-derived CM and selected the ten ACP candidates (designated P01, P02, P03, P04, P05, P06, P07, P08, P09, and P10) that were highly expressed in the peptide pool. After the prescreening of these candidates, we selected peptide P05 as an especially potent ACP for further characterization. P05 was derived from aldolase A (ALDOA), which is a glycolytic enzyme responsible forconverting fructose- 1 ,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. The levels of glycolytic enzymes, including ALDOA, are generally elevated in cancer cells, and they contribute to inducing the Warburg effect (i.e., most cancer cells produce energy predominantly not through the “usual” citric acid cycle and oxidative phosphorylation in the mitochondria as observed in normal cells, but through a less efficient process of 'aerobic glycolysis' consisting of a high level of glucose uptake and glycolysis followed by lactic acid fermentation taking place in the cytosol, not the mitochondria).

[0273] Accordingly, cancer cells preferentially use glycolysis rather than oxidative phosphorylation for energy production. Importantly, we have shown that extracellular ALDOA, which is enriched in iTSC-derived CM, acts as a tumor-suppressing protein in breast cancer. In this study, we presented that ACPs can be identified from iTSC-derived tumor-suppressing proteins, and ALDOA-derived P05 is a potential candidate for the treatment of OS.

[0274] Materials and Methods.

[0275] Cell Culture

[0276] Human OS cell lines such as MG63 and U2OS (86051601-1VL and 92022711- 1VL. Sigma. St. Louis, MO, USA), a patient-derived xenograft (PDX) TT2-77 OS xenolme, murine K7M2 osteosarcoma cancer cells (ATCC, Manassas, V A, USA), RAW264.7 preosteoclast cells (ATCC), and murine mesenchymal stem cells (MSCs) derived from the bone marrow of the C57BL / 6 strain were cultured in Dulbecco’s Modified Eagle Medium (DMEM). MC3T3-E1 osteoblasts (Sigma) were grown in aMEM (Gibco, Carlsbad, CA, USA), and human fetal osteoblast (hFOB) (ATCC) were grown in DMEM. The culture medium was supplemented with 10% fetal bovine serum and antibiotics (100 units / mL penicillin, and 100 pg / mL streptomycin; Life Technologies, Grand Island, NY, USA). Cells were maintained at 37°C and 5% CO2.

[0277] OS cells were treated with 10 peptides (P01 to P10, Genscript Biotech, Piscataway. NJ, USA) and chemotherapeutic agents, such as Taxol (#1097, Tocris Bioscience, Minneapolis, MN, USA), Doxorubicin (#2252, Tocris Bioscience), and Cisplatin (#2251, Tocris Bioscience). TT2 OS cells were also treated with human recombinant ALDOA protein (#MBS8248528. MyBioSource. San Diego, CA, USA). For in-vitro experiments. CM was prepared using low-speed centrifugation at 2,000 rpm for 10 minutes. The supernatants were further centrifuged at 4,000 rpm for 10 minutes and subjected to filtration with a 0.22-pm polyethersulfone membrane (Sigma).

[0278] MTT Assay and EdU Assay

[0279] A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT)-based metabolic activity assay was performed using -2,000 cells seeded in 96-well plates. Cells were incubated for two days with treatment agents. They were dyed with 0.5 mg / mL thiazolyl blue tetrazolium bromide (#M5655, Sigma) on day 4, and a mixed solution of isopropyl alcohol and hydrochloric acid was added at the ratio of 60: 1. Optical density for assessing metabolic activities was determined at 562 nm. A 5-ethynyl-2'-deoxyuridine (EdU)-based proliferation activity assay was performed using - 1,000 cells, which were seeded in 96-well plates. We employed a fluorescence-based cell proliferation kit (Click-iT™, EdU Alexa Fluor™ 488 Imaging Kit; Thermo Fisher, Waltham, MA, USA), using the procedure described by the manufacturer.

[0280] Scratch Assay

[0281] A wound-healing scratch assay was utilized to evaluate the two-dimensional migratory behavior of OS cells by determining the change in the scratch area. Approximately 3>< 103cells were seeded in 12-well plates on day 1. On day 2, a scratch was made on the cell layer with atip of a plastic pipette. Cells were washed with Dulbecco’s Modified Eagle Medium (DMEM) to remove floating cells. Images of cell-free zones were taken at 0 hours and 24 hours with a light microscope (40 x), and the areas of the cell-free zone were quantified by ImageJ software.

[0282] Transwell® Invasion Assay

[0283] The invasion capacity’ of tumor cells was determined using a 12-well plate and Transwell® chambers (#353182, Thermo Fisher) with 8-pm pore size. The chambers were coated with 300 pL Matrigel® (100 pg / mL), and 500 pL of the serum-free medium was added. The chamber was washed three times with the serum- free medium. Approximately 7 x 104cells in 300 pL serum-free DMEM were placed in the upper chamber, and 800 pL CM was added to the lower chamber. Cells, which invaded the lower side of the membrane, were fixed, and stained with methanol and crystal violet. Five randomly chosen images were taken with an inverted optical microscope (100x), and the average number of stained cells, which represented the invasion capacity, was determined.

[0284] Western Blot Analysis

[0285] Cells were lysed with a radioimmunoprecipitation assay (RIP A) lysis buffer (#sc-24948, Santa Cruz Biotechnology7, Dallas, TX, USA) with protease inhibitors (#PIA32963, Thermo Fisher), and phosphatase inhibitors (#2006643, Cal-biochem, Billerica, MA, USA). Proteins were separated by 10%- 15% sodium dodecyl sulfate (SDS) gel (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to a poly vinylidene difluoride membrane (#IPVH00010, Millipore, Billerica, MA, USA). The membrane was incubated with the primary antibodies.followed by incubation with secondary antibodies (#7074S / 7076S, Cell Signaling. Danvers, MA, USA). We used antibodies against epidermal growth factor receptor (EGFR). Aldolase A (ALDO A), Snail, p-Src, Src, cleaved caspase 3, caspase 3, IL-1 p, IL-6 (Cell Signaling), and M- CSF, OPN (Santa Cruz Biotechnology), with -actin (Sigma) as a control. The protein level was determined using a SuperSignal west femto maximum sensitivity substrate (#PI34096, Thermo Fisher), and a luminescent image analyzer (#LAS-3000, Fuji Film, Tokyo, Japan) was used to quantify signal intensities.

[0286] Plasmid Transfection and RNA Interference

[0287] The overexpression of ALDOA was conducted by transfecting plasmids (#109854; Addgene. Cambridge, MA, USA), while blank plasmids (FLAG-HA-pcDNA3.1; Addgene) were used as a control. RNA interference was conducted using siRNA specific to EGFR (#AM16708; Thermo Fisher) with anegative siRNA (Silencer Select #1, Thermo Fisher) as a nonspecific control using the procedure previously described.

[0288] Immunoprecipitation

[0289] Immunoprecipitation was conducted with an immunoprecipitation starter pack kit (#45-000-369, Cytiva, Marlborough, MA, USA), using the procedure according to the manufacturer. Protein samples were pretreated with agarose beads conjugated with protein A and rabbit IgG, followed by overnight immunoprecipitation with the beads conjugated with anti- EGFR or ALDOA antibodies. The beads were collected by centrifugation, washed three times with phosphate buffer solution (PBS), and resuspended for Western blotting. Western blotting was conducted using antibodies against EGFR and ALDOA (Cell Signaling).

[0290] Bone Culture

[0291] A pair of femora were collected from C57BL / 6 female mice (~10 weeks of age), and connective tissues around the bone were removed. The femora were cut in half at the middiaphysis, and a hole was generated at the other side with a 25-gauge needle. K7M2 OS cancer cells (2.5 / I O5cells) were resuspended in 10 pL of culture medium, and they were injected into the bone marrow cavity through the open end of the diaphysis. The bone samples were placed into a petri dish and cultured in 2 mL of DMEM with 10% fetal bovine serum (FBS) and 1% antibiotics at 37 °C and 5% CO2. Half of the culture media was replaced daily with fresh medium. After 2 weeks, the bone samples were used for Western blot analysis.

[0292] Osteoclast Differentiation Assay

[0293] The differentiation assay of RAW264.7 pre-osteoclasts was performed in a 12- well plate. During the 6-day incubation of pre-osteoclast cells in 40 ng / mL of Receptor activatorof nuclear factor kappa-B ligand (RANKL), the CM was exchanged once on day 4. Adherent cells were fixed and stained with a tartrate-resistant acid phosphate (TRAP)-staining kit (Sigma), according to the manufacturer’s instructions. TRAP-positive multinucleated cells (> 3 nuclei) were identified as mature osteoclasts.

[0294] Molecular Docking Analysis

[0295] To evaluate interactions between EGFR and ALDOA, as well as EGFR and P05, a ZDOCK program (ver. 2016, Discovery Studio, San Diego. CA, USA) was employed. All possible binding poses were considered in the translational and rotational space between EGFR and ALDOA or P05, and each pose was evaluated by determining the energy-based ZDOCK scoring system. Hydrogen-bonding interactions were predicted for the EGFR-ALDOA complex and EGFR-P05 complex.

[0296] Statistical Analysis

[0297] For cell-based experiments, three or four independent experiments were conducted, and data were expressed as mean ± S.D. Statistical significance was evaluated using a one-way analysis of variance (ANOVA). Post hoc statistical comparisons with control groups were performed using Bonferroni correction with statistical significance at p < 0.05. The single and double asterisks in the figures indicate p < 0.05 and p < 0.01, respectively.

[0298] Results.

[0299] Reduction In Viability, and Motility of OS Cells by P02, P04, andP05

[0300] We first examined the ten anti-tumor peptide candidates (P01 to P10). They were selected from engineered MSC-derived conditioned medium (CM) that was enriched with antitumor proteins (Table 4).

[0301] The peptide candidates were derived from the trypsin-digested fragments of all proteins in the CM and their anticancer capability was evaluated using three OS cell lines (U2OS, TT2, and MG63). While the reduction of MTT-based cell viability in 2 days was observed with the peptides at 50 pg / ml except for peptide P06, the peptides (P02, P04, and P05) were the top three in the average reduction in the viability (Table 5).

[0302] Among these three peptides, P05 presented the most significant anti-tumor effects, particularly, on the MTT-based viability and scratch-based cell motility for TT2 OS cells (Figs. 1 1A-1 IB). Importantly, these three peptides did not reduce the viability or motility of MSCs and hFOBs (Figs. 11C-11D, Figs. 18A-18B), indicating their cancer-selective inhibitory actions.

[0303] Reduction in Proliferation, and Invasion of OS Cells by P05

[0304] The reduction in MTT viability was dose-dependent for P02, P04, and P05 (Fig. 12 A), and combinatorial use of these peptides presented a combinatorial effect on the reduction in cell motility but not on the reduction in cell viability (Figs. 12B-12C). Of note, P02 was derived from Rho GDP dissociation inhibitor alpha (ARHGDIA), while P04 and P05 were part of Aldolase A (ALDOA), a glycolytic enzyme. Besides viability and motility, P05 decreased the EdU-based proliferation and Transwell® invasion of TT2 OS cells (Figs. 13A-13B). Furthermore. P05 decreased the scratch-based motility of U2OS and MG63 OS cells and the EdU-based proliferation of MG63 cells (Figs. 19A-19C). Also, the application of P05 to TT2 OS cells down regulated oncoproteins such as p-Src, EGFR, and Snail, while it acted as a cytotoxic agent by elevating cleaved caspase 3, an apoptotic marker (Fig. 13C). In MG63 OS cells. P05 also decreased the level of EGFR, p-Src, and Snail (Fig. 19D). Hereafter, we mainly examined the inhibitory action of P05 because of its superior anti-tumor capabilities.

[0305] Combined Anti-Tumor Effects With Chemotherapeutic Agents

[0306] We next examined the effects of the simultaneous application of P05 with three representative chemotherapeutic agents, such as Cisplatin, Doxorubicin, and Taxol. Cisplatin and Doxorubicin are two of the three agents in the standard-of-care chemotherapy for OS. while Taxol is the most well-known natural-source cancer drug. The result revealed that the application of P05 contributed to lowering the ICso of the selected agents in MTT-based viability' of TT2 OS cells (Fig. 13D). With the application of P05 at 25 pg / ml, the ICso value was lowered from 7.4 pM to 4.7 pM (Cisplatin). 2.6 pM to 0.9 pM (Doxorubicin), and 0.5 pM to 0.1 pM (Taxol).

[0307] Anti-Tumor Effects On Breast Cancer Cells by P02, P04, and P05

[0308] Besides the anti-OS actions, we observed that peptides P02, P04, and P05 suppressed the progression of breast cancer cells. The application of 25 pg / ml of P02, P04. and P05 significantly reduced MTT-based viability and scratch-based motility of MDA-MB-231 breast cancer cells (Figs. 14A-14B). Furthermore, P05 significantly reduced Transwell® invasion and EdU-based proliferation of MDA-MB-231 cells (Figs. 14C-14D). It also reduced the expression of EGFR. p-Src, and Snail (Fig. 14E).

[0309] Generation of iTSCs by the Overexpression of ALDOA in MSCs

[0310] Thus far, we show that P05, which is derived from ALDOA, can act as an ACP for OS and breast cancer. We next evaluated the effect of ALDOA, a parental protein of P04 and P05, on OS cells and MSCs. As reported in our previous studies, most tumor-suppressing proteins, which were enriched in iTSC CM such as Moesin, Enolase 1, etc., presented a doublesided action as an oncoprotein intracellularly and a tumor suppressor extracellularly. Here, wealso observed the same site-dependent action of ALDOA. When ALDOA was overexpressed in TT2 and MG63 OS cells, it acted as an oncoprotein and stimulated the viability of OS cells (Fig. 15 A). However, when ALDOA was transfected into MSCs, ALDOA-ov erexpressing MSC- derived CM presented tumor-suppressing capabilities. This CM reduced viability, proliferation, motility, and invasion of TT2 OS cells (Figs. 15B-15F), as well as MG63 OS cells (Figs. 20A- 20D).

[0311] Interaction Between ALDOA and EGFR

[0312] In the IntAct molecular interaction database (EMBL-EBI), the interaction of ALDOA with EGFR was predicted (Figs. 21A-21C). Consistently, a protein docking program, ZDOCK, predicted the potential binding of ALDOA with EGFR, including 22 possible interactions in the range of 1.9 to 3.7 A (Fig. 16A, Table 6).

[0313] Also, the possible binding of P05 with EGFR was shown with 10 putative hydrogen bondings at 1.9 A to 3.6 A (Fig. 16B, Table 7). An immunoprecipitation assay usinga protein lysate from TT2 OS cells showed that ALDOA was co-immunoprecipitated with EGFR, and reciprocally EGFR was co-immunoprecipitated with ALDOA (Figs. 16C-16D). Consistently, silencing of EGFR with its specific siRNA suppressed ALDOA-induced reduction in cell viability (Fig. 16E). Collectively, ALDOA is a double-edged protein that acts as an oncoprotein intracellularly, as well as a tumor suppressor extracellularly. While the potential binding of P05 to EGFR was predicted, the interactions between ALDOA and EGFR (ZDOCK score of 27.78) were predicted stronger than those between P05 and EGFR (ZDOCK score of 15.76).

[0314] Inhibition of Osteoclast Maturation by P05

[0315] Bone-resorbing osteoclasts play a critical role in bone degradation in OS. Besides inhibiting the progression of OS cells, we examined the effects of P05 on the development of osteoclasts. The result showed that the differentiation of RANKL-stimulated osteoclasts was blocked and the number of multi-nucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts (more than 3 nuclei) was significantly lowered by the application of P05 at 25 pg / ml (Fig. 17A). By contrast, no significant change in the viability of MC3T3-E1 osteoblast cells was detected in response to P05, with a slight increase in type I collagen (COLI) and alkaline phosphatase (ALP) (Figs. 21B-21C).

[0316] Reduction of Oncoproteins by P05 in the Ex-Vivo Bone Culture

[0317] Given the anticancer role of P05 in OS cells, we lastly examined its tumorsuppressing effect using an ex-vivo bone culture. K7M2 OS cell-inoculated femora were cultured in the presence and absence of 25 pg / mL P05 for 2 weeks, and the expression ofoncoproteins was evaluated. The result revealed that the administration of P05 significantly reduced the levels of EGFR. macrophage colony-stimulating factor (M-CSF), osteopontin (OPN), interleukin 1 beta (IL-1 P), interleukin 6 (IL-6), and phosphorylated Src (Fig. 17B).

[0318] Discussion.

[0319] This Example presented that peptide P05 acted as an effective ACP for OS cells as well as breast cancer cells. P05 is derived from a glycolytic enzyme, ALDOA, having an amino acid sequence of ADDGRPFPQVIK (1,342 Da) (SEQ ID NO.: 5) with an isoelectric point of 7.0. The application of P05 inhibited the viability, proliferation, motility, and invasion of human OS cell lines. It also downregulated the expression of oncogenic proteins such as Src, Snail, and EGFR and induced apoptosis by elevating the level of cleaved caspase 3. P04 was also derived from ALDOA and acted as another ACP (2, 107 Da for IGEHTPSSLAIMENANVLAR) (SEQ ID NO.: 4) with an isoelectric point of 5.3. Most ACPs are positively charged since cancer cells have in general negative surface charges because of the glycolysis-driven secretion of lactic acid. Interestingly, unlike most ACPs, the net charge of P04 and P05 is negative at a neutral pH. The result indicates that the selective interaction of P04 and P05 with OS cells is not mediated by non-specific electrostatic force.

[0320] We observed a double-sided role of ALDOA, depending on its acting site in the intracellular and extracellular domains. Similar to atypical tumor-suppressing proteins, such as Moesin and Enolase 1, its overexpression in tumor cells elevated their MTT-based viability. However, ALDOA-overexpressing MSCs generated tumor-suppressing CM, and extracellular ALDOA acted as a tumor suppressor. The protein docking analysis and immunoprecipitation assay supported the interaction of ALDOA with EGFR. The overexpression of EGFR promoted the growth and motility of OS cells, while its deletion suppressed the proliferation, migration, and invasion of OS cells in vitro and tumor formation in vivo. The levels of phospho- Akt and phospho-ERK were decreased by EGFR knockdown but increased as a result of EGFR overexpression, supporting a possible involvement of PI3K / Akt and ERK pathways in the effects of EGFR on OS cells.

[0321] Deep learning-based analyses present a list of selection criteria for predicting effective ACPs. The criteria include (a) enrichment of glycine, alanine, leucine, phenylalanine, tryptophan, and lysine residues, (b) high frequency of the positively charged residue lysine and low' frequency of the negatively charged residues of aspartic acid and glutamic acid, (c) a net positive charge with a high isoelectric point, and (d) frequent appearance of specific pairing. The net charge at pH 7 was 0 (P05) and -0.91 (P04). According to one of the prediction softwareiDACP, P04 and P05 were predicted as non-ACPs with a score of 0.039 and 0.074, respectively, in which the score range was 0 (most likely non-ACP) to 1 (most likely ACP). The non-ACP prediction by iDACP indicates the difficulty in predicting effective ACPs.

[0322] By way of theory, the proposed mechanism of P05’s action is illustrated in Fig. 17C. The overexpression of ALDOA in OS cells promoted their viability, but ALDOA- overexpressing MSC CM, as well as extracellular ALDOA, suppressed the progression of OS cells. ALDOA-derived P05 also inhibited the proliferation, migration, and invasion of OS cells. The combinatorial effect of P05 and chemotherapeutic agents is an advantage for clinical applications. Patients do not have to eliminate the current standard-of-care treatment. The simultaneous application of P05 significantly reduced IC50 of Cisplatin, Doxorubicin, and Taxol. The combinatorial therapeutic action would reduce the toxic effects of the chemotherapeutic agents by reducing their dosage. Besides the suppression of OS progression, P05 was effective in blocking the differentiation of bone-resorbing osteoclasts.

[0323] Conclusions.

[0324] This Example demonstrated that P05 is a candidate for anti-OS APC that suppressed the viability, motility, and invasion capability of three OS cell lines. P05 can be applied with existing chemotherapeutic agents, and it significantly reduced the IC50 of each of the three tested agents. The anticancer action of P05 is not based on nonspecific electrostatic interactions with negatively charged cancer cells. Further studies are recommended to determine its anti-cancer mechanism, including possible interaction with EGFR.Example 3: P18: Novel Anticancer Peptide from Induced Tumor-Suppressing Cells Targeting Breast Cancer and Bone Metastasis.

[0325] The skeletal system is a common site for metastasis from breast cancer. Induced tumor-suppressing cells (iTSCs) were developed capable of secreting a set of tumor-suppressing proteins. In this example, the possibility was examined of identifying anticancer peptides (ACPs) from trypsin-digested protein fragments derived from iTSC proteomes.

[0326] Background.

[0327] Breast cancer, a malignancy originating in breast tissue, initiates abnormal cell proliferation within breast glandular or ductal tissue, leading to the formation of a cancerous mass. Key risk factors include age, family medical history, genetics, hormone levels, lifestyle choices, and various environmental and physiological elements. Early detection and intervention are imperative for the global prevalence of this disease among women due to the formidable challenge posed by metastasized breast cancer. Treatment modalities includesurgical resection, radiotherapy, chemotherapy, hormone therapy, and targeted therapy. Notably, bone metastasis is common, yet there is no established curative treatment strategy. Ongoing research is dedicated to advancing approaches aimed at preventing bone loss and enhancing the survival rates of individuals with bone-metastasized breast cancer.

[0328] This example is aimed at the formulation and testing of potent anticancer peptides (ACPs) for the treatment of bone metastasis associated with breast cancer. ACPs offer a promising avenue for cancer treatment due to their nonbiological processes in synthesis and quality control, distinguishing them from other cell- and protein-based agents. Typically, comprising basic and hydrophobic residues with positive charges, ACPs are believed to align with the negatively charged nature of most cancer cells. Despite the availability of databases such as CancerPPD, ImmunoPeptidome, and PeptideCutter for designing ACP candidates and predicting their tumor-binding epitopes, the effective design of ACPs remains challenging. A particular difficulty lies in identifying specific targets and differentiating cancer cells from normal cells.

[0329] A unique approach of the present example involved the derivation of ACP candidates from tumor-suppressing proteins secreted by induced tumor-suppressing cells (iTSCs). Notably, previously demonstrated were the generation of iTSCs and their tumorsuppressive conditioned medium (CM) from both cancer and non-cancer cells through the modulation of diverse signaling pathways. Intriguingly, iTSCs can be generated by the overexpression of cMyc or Oct4, in contrast to induced pluripotent stem cells (iPSCs), which require the overexpression of four transcription factors: cMyc, Oct4, Klf4, and Sox2. Prior research has also established that the CM derived from mesenchymal stem cells (MSCs) overexpressing Akt and -catenin selectively inhibits tumor cell proliferation and invasion. Additionally, the induction of iTSCs was found to be effective through the activation of Protein kinase A (PKA) signaling in Jurkat T lymphocytes or the inhibition of AMP-activated protein kinase (AMPK) signaling in human peripheral blood mononuclear cells (PBMCs). An intriguing question is whether ACPs can be developed from a secretome of iTSCs.

[0330] In earlier ACP investigations, the efficacy was highlighted of a unique pair of ACPs, namely P04 for pancreatic ductal adenocarcinoma and P05 for osteosarcoma (OS). Both P04 and P05 were derived from aldolase A, a glycolytic enzyme known to induce the Warburg effect in cancer cells. In this current example, a screening of ACPs from a library of fragments of iTSC-derived tumor-suppressing proteins revealed the potent antitumor capabilities of anew ACP. Pl 8, for breast cancer cell lines. P18 is derived from ARHGDIA, a Rho GDP dissociation inhibitor alpha. This example demonstrates the effectiveness of P18 in safeguarding bone frombreast cancer cells, by exploring the role of GTPase in breast cancer and its associated impact on bone loss.

[0331] Materials and Methods.

[0332] Cell Culture

[0333] Two human triple-negative breast tumor cell lines, MDA-MB-231 and MDA- MB-436 (ATCC, Manassas. VA, USA), were cultured in RPMI-1640 (Gibco, Carlsbad, CA, USA). Eight different tumor and bone cell types, cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Coming Inc., Coming, NY, USA), included MCF7 human estrogen receptor-positive breast cancer cells (ATCC), 4T1.2 mouse mammary tumor cells (obtained from Dr. Robin Anderson at Peter MacCallum Cancer Centre, Melbourne, Australia), PANCI (ATCC) and PANCI 98 (sourced from Indiana University Simon Comprehensive Cancer Center, Indianapolis, IN, USA) pancreatic cancer cells, a xenograft TT2-77 osteosarcoma cells, MG63 osteosarcoma cells (Sigma, St. Louis, MO, USA), RAW264.7 pre-osteoclast cells (ATCC), and murine MSCs that were derived from the bone marrow of C57BL / 6 mice. MC3T3- E1 osteoblasts (Sigma) were grown in aMEM (Gibco, Carlsbad, CA, USA). The culture medium was supplemented with 10% fetal bovine serum and antibiotics (100 units / mL penicillin, and 100 pg / mL streptomycin; Life Technologies, Grand Island, NY, USA). Tumor cells were treated with chemotherapeutic agents, such as Cisplatin (3259, Tocris Bioscience, Minneapolis, MN, USA) and Taxol (3257, Tocris Bioscience), as well as human recombinant ARHGDIA protein (MBS8248528, MyBioSource, San Diego, CA. USA).

[0334] Peptides Pl 1 to P22, and P 18 Analogs

[0335] As ACP candidates, 12 preptides were initially examined(Pl 1 to P22, Genscript Biotech, Piscataway. NJ, USA) (Table 8). P18 was selected for further analysis in this study. To evaluate whether Pl 8 is located at the effective location along ARHGDIA protein, we examined Pl 8-N5 and Pl 8-C5, which were shifted along ARHDIA towards N-terminus and C- terminus, respectively, by 5 amino acid residues. To further examine the role of specific residues of Pl 8, the eleven Pl 8 analogs, Pl 8-Al to P18-A11, were evaluated by replacing each of the eleven amino acids of Pl 8 with alanine. Alanine is a non-polar amino acid with uncharged and hydrophobic side chains, which may stabilize P18 but not enhance interactions with binding partners. To improve the efficacy of P18, we further generated the three P18 analogs by modifying N and C-termini with acetyl and amino groups (AC-PI8-NH2 and PI8-NH2), together with testing a shorter version, P18S, with the same terminal modification (AC-P18S- NH2).

[0336] MTT and EdU Assays

[0337] Using 96-well plates with -2,000 cells seeded in each well, an MTT-based metabolic activity was conducted. Cells were incubated for two days with treatment agents, and on day 4 they were dyed with 0.5 mg / mL thiazolyl blue tetrazolium bromide (M5655, Sigma). Optical density for assessing metabolic activities was determined at 562 nm. EdU-based proliferation activity was evaluated using 96-well plates with -1,000 cells in each well. A fluorescence-based cell proliferation kit (Click-iT™, EdU Alexa Fluor™ 488 Imaging Kit; Thermo Fisher, Waltham, MA, USA) was employed, using the procedure described by the manufacturer.

[0338] Scratch Assay

[0339] To evaluate the two-dimensional motility of cancer cells, a wound-healing scratch assay was utilized and the change in the scratch area was determined. On day 1 , - 3 x 105cells were seeded in 12-well plates, and on day 2 a scratch was made on the cell layer with the tip of a plastic pipette. Using a light microscope (40 x), cell-free zones were imaged at 0 h and 24 h, and the alteration in their areas was quantified by ImageJ public-domain image processing software.

[0340] Transwell® Invasion Assay

[0341] In response to ACPs, the invasion capacity of cancer cells was determined using 8-pm pore Transwell® chambers (353182. Thermo Fisher) in a 12-well plate. The chambers were coated with 300 pL Matrigel™ (100 pg / mL), and 500 pL of the serum-free medium wasadded. The chamber was washed three times with the serum-free medium. Approximately 7x 104cells in 300 pL serum-free DMEM were placed in the upper chamber, and 800 LIL medium was added to the lower chamber. Cells, which moved to the lower side of the membrane, were fixed and stained with methanol and crystal violet. Five randomly chosen images were taken with an inverted optical microscope (100x), and the average number of stained cells, which represented the invasion capacity, was determined.

[0342] Western Blot Analysis

[0343] Cells were lysed with a Radio-Immunoprecipitation Assay (RIP A) lysis buffer (sc-24948, Santa Cruz Biotech, Dallas, TX, USA) with protease inhibitors (PIA32963, Thermo Fisher) and phosphatase inhibitors (2006643, Cal-biochem, Billerica, MA, USA). Proteins were separated by 10%— 15% sodium dodecyl sulfate (SDS) gels (Bio-Rad Laboratories. Hercules, CA, USA) and transferred to a poly vinylidene difluoride membrane (IPVH00010, Millipore, Billerica, MA, USA). The membrane was incubated with the primary antibodies, followed by incubation with secondary' antibodies (7074S / 7076S, Cell Signaling. Danvers, MA, USA). Antibodies were used against Snail, p-Src, Src, cleaved caspase 3, caspase 3. RANKL, Cdc42, and RhoA (Cell Signaling) with (3-actin (Sigma) as a control. The protein level was determined using a SuperSignal west femto maximum sensitivity substrate (PI34096, Thermo Fisher) and a luminescent image analyzer (LAS-3000, Fuji Film, Tokyo, Japan).

[0344] Fluorescence Resonance Energy Transfer (FRET)-Based GTPase Activity Assay

[0345] The three FRET GTPase biosensors were utilized to monitor the activities of Rael , Cdc42, and RhoA in response to ACPs. GTPase activity was estimated based on the emission ratio of yellow' fluorescent protein (YFP) to cyan fluorescent protein (CFP), using a Nikon Ti-E inverted microscope with an electron-multiplying charge-coupled device (EMCCD) camera (Photometries), a filter wheel controller (Sutter Instruments), and a Nikon Perfect Focus System. To reduce photobleaching, an Intensilight™ fluorescence illuminator (Nikon) was employed with a neutral density 32 filter (3% transmittance). The images were captured with a 40 x (0.75 numerical aperture) objective, and they were scaled utilizing the color bar, as previously published in Wan et al., Rael and Cdc42 GTPases regulate shear stress-driven beta- catenin signaling in osteoblasts. Biochem Biophys Res Commun 2013, 433. 502-507.

[0346] Ex-Vivo Cancer Tissue Assay

[0347] The usage of human triple-negative breast cancer tissue was approved by the Indiana University Institutional Review' Board (1911155674), and the tissues were received from Indiana University Simon Comprehensive Cancer Center Tissue Procurement Core. A freshly isolated breast cancer tissue (~10 mg) was manually fragmented with a scalpel into smallpieces (0.5 ~ 0.8 mm in length), which were grown in DMEM with 10% FBS and antibiotics for a day. The described CM was then added for 4 additional days, and a change in the fragment size was determined.

[0348] Bone Microenvironment Assay

[0349] A pair of femora were collected from C57BL / 6 female mice (-10 weeks of age), and connective tissues around the bone were removed. The femora were cut in half at the middiaphysis, and a hole was generated at the end of the sample with a 25-gauge needle. 4T1.2 mammary tumor cells (2.5 x 105cells) were resuspended in 10 pL of culture medium, and they were injected into the bone marrow cavity through the open end of the diaphysis. The bone samples were placed into a petri dish and cultured in 1.5 mL of DMEM with 10% FBS and 1% antibiotics at 37 °C and 5% CO2. Half of the culture media was replaced daily with fresh medium. After 2 weeks, the bone samples were harvested and used for Western blot analysis.

[0350] Molecular Docking Analysis

[0351] Molecular docking analysis was conducted to evaluate the interactions between Rho GDP dissociation inhibitor (ARHGDIA) and small GTPase (Cdc42), as well as a fragment of ARHGDIA. Pl 8, and Cdc42. The structures of ARHGDIA and Cdc42 were obtained from the Protein Data Bank PDB database, while the P18 structure was assumed as the fragment structure of amino acids 142-152 in ARHGDIA protein. The GRAMM (Global RAnge Molecular Matching) molecular docking software was employed to predict the most stable relative configurations of the two interacting molecules. The binding mode and hydrogen bonds were selected with the highest score using the PDBePISA (Protein Data Bank in Europe, Proteins, Interfaces, Structures, and Assemblies) database with PyMOL Educational software (Version 2.3.2).

[0352] Statistical Analysis

[0353] For cell-based experiments, three or four independent experiments were conducted, and data were expressed as mean ± S.D. Statistical significance w as evaluated using a one-way analysis of variance (ANOVA). Post-hoc statistical comparisons with control groups were performed using Bonferroni correction with statistical significance at p < 0.05. The single and double asterisks in the figures indicate p < 0.05 and p < 0.01. respectively.

[0354] Results.

[0355] Anticancer Effects ofP18 On Breast Cancer Cells

[0356] In our initial assessment, tw elve ACP candidates (Pl 1 to P22) were screened and identified from the conditioned medium obtained from MSC-driven iTSCs (Table 8, supra). These peptides were derived from trypsin-digested fragments originating from a group ofproteins within the iTSC CM. Among all the peptides tested, P18 exhibited a significant reduction on average in cell viability (Table 9). Subsequently, the investigation focused on Pl 8 and evaluated its anti-tumor effects, mostly on breast cancer cell lines and tissues. The decrease in MTT viability of MDA-MB-231, MDA-MB-436, and MCF7 cells was found to be dosedependent (Fig. 22a). Scratch assays demonstrated that Pl 8 effectively reduced the motility of MDA-MB-231 cells (Fig. lb), while the EdU assay and Transwell® invasion assay revealed that Pl 8 decreased their proliferation and invasive properties (Fig. 22c&d). Consistently, the application of P18 to MDA-MB-436 cells inhibited their scratch-based mobility (Fig. 22e) as well as their EdU-based proliferation and transwell-based invasive properties (Fig. 22f&g).

[0357] Anti-Tumor Effect of P18 On Other Tumor Cell Lines

[0358] The investigation revealed that Pl 8 acted as an ACP not only in breast cancer cells but also in pancreatic ductal adenocarcinoma (PDAC) and osteosarcoma cells. Application of 25 pg / ml of Pl 8 significantly decreased the MTT-based viability' of two PDAC cell lines, PANCI and PAN198 (Fig. 23a), as well as TT2 osteosarcoma cell line (Fig. 23b). Moreover, this treatment consistently reduced both the MTT-based viability and scratch-based mobility of 4T1.2 mammary tumor cells (Figs. 23c&d).

[0359] Enhanced Anti-Tumor Effects In Combination With Chemotherapeutic Agents

[0360] Given the role of Pl 8 in tumor suppression, we next examined its efficacy with the application of chemotherapeutic agents. The result showed that the application of Pl 8 resulted in a significant reduction of the ICso value for Cisplatin, from 1.2 pM to 0.5 pM, and for Taxol, from 0.7 pM to 0.3 pM, as determined by the MTT-based viability assay on MDA- MB-231 cells (Fig. 24a).

[0361] To further evaluate the efficacy of P18, we exposed it to recently acquired fragments of human triple-negative breast cancer tissue. These tissue fragments were partitioned into two cohorts: one was cultivated in a conventional growth medium, while the other was treated with P18. Throughout four days, we monitored changes in their morphological characteristics. The results demonstrated a significant decrease in the size of ex-vivo breast tissue fragments during the four-day P 18 treatment (Fig. 24b). Moreover, our findings suggested that the concurrent administration of Pl 8 displayed enhanced effectiveness in contrast to theindividual use of Cisplatin or Taxol, leading to a more pronounced reduction in the size of cancer tissues after 48 and 96-h treatment (Fig. 24c. Fig. 30a).

[0362] Combinatorial Effects With Other ACPs As Well As Anti-Osteolytic Effects

[0363] Also examined was a combined effect with other ACPs. The reduction in MTT viability was observed with other ACPs such as P04 and P05, and combining these peptides with P18 resulted in an additive effect on reducing cell viability (Fig. 25a). When applied to MDA-MB-231 and MDA-MB-436 breast cancer cells, Pl 8 downregulaled oncoproteins like p- Src and Snail, while simultaneously acting as a cytotoxic agent by increasing the levels of cleaved caspase 3, an apoptotic marker (Fig. 25b).

[0364] Beyond its impact on breast cancer cell progression, we also investigated the effects of P18 on the development of bone-resorbing osteoclasts. The results revealed that Pl 8, at a concentration of 25 pg / ml. inhibited the differentiation and maturation of RANKL- stimulated osteoclasts. The application of Pl 8 significantly reduced the number of multinucleated TRAP -positive osteoclasts (with more than 3 nuclei) (Fig. 25c).

[0365] P 18-Containing Protein ARHGDIA and Its Binding Partners

[0366] As Pl 8 is a fragment of ARHGDIA. known as Rho GDP-dissociation inhibitor 1, it can bind to Rho GTPases, thereby preventing their binding to effector molecules. This action maintains these molecules in an inactive GDP-bound state, crucially regulating intracellular signaling and various cellular functions like cytoskeletal remodeling, cell migration, differentiation, and proliferation. An extensive analysis of the TCGA database uncovered a correlation between elevated transcript levels of ARHGDIA across all cancers and compromised survival rates (Fig. 26a).

[0367] As a protein that is enriched in the secretome of iTSCs, ARHGIDIA acts as a tumor-suppressing protein. Consistently, the use of recombinant human ARHGDIA protein (rhARHGDIA) resulted in a significant reduction in MTT-based cell viability (Fig. 26b). Notably, the IntAct molecular interaction database predicted interactions of ARHGDIA with RhoA and Cdc42 (Fig. 26c). Additionally, when Pl 8 was applied to MDA-MB-231 and MDA- MB-436 breast cancer cells, a slight upregulation of RhoA and Cdc42 was observed (Fig. 26d). This suggests that the effect of P l 8 involves inhibiting the activity of Cdc42 and RhoA rather than causing the downregulation of their expression.

[0368] Effects ofP18 On GTPase Activities of Rael. RhoA, and Cdc42

[0369] The experiment next investigated the role of P 18 in regulating the activity of Rho family GTPases. MDA-MB-231 breast cancer cells were transfected with Rael, Cdc42, or RhoA biosensors and treated with Pl 8 at a concentration of 25 pg / mL for 3 hours. The GTPaseactivity was assessed by measuring the emission ratio of the biosensor within individual live cells. The findings revealed a significant decrease in the activities of RhoA and Cdc42 in response to Pl 8, while no significant change was observed in Rael activity (Figs. 27a-27c). Moreover, between RhoA and Cdc42, the activity of Cdc42 showed a more pronounced decrease compared to RhoA, suggesting that the inhibition of Cdc42 activation may hold greater significance for Pl 8 as an ACP.

[0370] Interaction Between ARHGDIA and Cdc42

[0371] The IntAct molecular interaction database highlighted a potential interaction between ARHGDIA and Cdc42. To evaluate this connection, protein-protein docking was performed, resulting in a score of 478, suggesting probable binding between ARHGDIA and Cdc42. This analysis revealed 14 possible interactions (hydrogen bond distances ranging from 2.1 to 3.7 A) (Table 10), with specific amino acid positions, such as GLU 87-LYS 131 and ARG 152-GLU 91, showing hydrogen bonding. Additionally, this investigation demonstrated the probable binding of P 18 to Cdc42, unveiling seven putative hydrogen bonds in the range of 2.7 to 3.7 A (Fig. 28a-28b, Table 11).

[0372] Next, improvement of the antitumor activity of Pl 8 was sought by shifting the fragment location along the ARHGDIA protein, altering amino acid residues, and / or modifying N and C-termini. Initially, it was sought to ascertain whether Pl 8 represented the most effective segment within the ARHGDIA protein. To explore this, the Pl 8 position was modified by shifting the overall sequence by 5 amino acids towards both the N- and C-termini, resulting in P18-N5 and P18-C5 variants. Metabolic assays based on MTT confirmed that the original Pl 8 retained the strongest tumor-suppressive effect among the four cancer cell lines tested (Figs. 31a&b). To pinpoint which specific amino acid within Pl 8 played a pivotal role, an alanine residue was substituted with each of the eleven amino acids present in Pl 8, generating 1 1 distinct P18 analogs. Subsequent MTT-based metabolic assays reaffirmed that the original P18 exhibited the most potent tumor-suppressive effect in MDA-MB-231 and MDA-MB-436 breast cancer cell lines, as well as in TT2 OS and PANCI PDAC cells (Fig. 32).

[0373] MTT-based metabolic assays showed that Pl 8 modified by N-terminal acetylation and C-terminal amidation (AC-PI8-NH2) exhibited stronger tumor suppressor effects than the original P18 on three breast cancer cell lines (Figs. 28c&d). Similarly, this P18 analog exhibited stronger tumor suppressor effects than the original P18 on 4T1.2 mammary tumor cells, TT2 OS cells, as well as PANCI and PAN198 PDAC cell lines (Figs. 33a&b). Notably, the MTT-based metabolic assays showed that the C-terminal amidated Pl 8 alone did not improve the tumor suppressor effect (Figs. 34a-34d). The illustration of Pl 8 analogs is shown in Fig. 28e.

[0374] Reduction of Oncoproteins By P18 In the Ex-Vivo Bone Culture

[0375] Considering the anti-cancer effects of Pl 8 observed in breast cancer cells, we further explored its tumor-suppressive potential using ex-vivo bone cultures. Illustrated in theoperational schematic, femurs inoculated with 4T1.2 mammary tumor cells were cultured for 2 weeks in the presence and absence of 25 pg / mL Pl 8, after which they were assessed for oncoprotein expression. Upon administration of Pl 8, the results revealed a significant reduction in the levels of p-Src in oncogenic signaling, Snail in EMT stimulation, RhoA as GTPase, p- Akt in PI3K signaling, and RANKL in osteoclast development. Conversely, an upregulation was observed in the levels of c-Cas 3, Cdc42, and alkaline phosphatase (ALP) (Fig. 29a). A proposed mechanism outlining the action of P 18 is depicted in Fig. 29b.

[0376] Discussion.

[0377] In this study, we demonstrated that Pl 8 inhibited the progression of cancer cell lines including breast cancer, pancreatic cancer, and osteosarcoma, as well as breast cancer tissue fragments, without significantly affecting MSCs. The combination of Pl 8 with chemotherapeutic agents such as Cisplatin and Taxol dramatically reduced the drug's half- inhibitory concentration (ICso), as well as the combination with other ACPs such as P04 and P05. Western blot results showed that the application of Pl 8 decreased the expression of protooncoproteins while increasing the expression of the apoptosis-associated protein c-Cas 3 in MDA-MB-231 and MDA-MB-436 breast cancer cell lines. Pl 8 inhibited the GTPase activity of RhoA and Cdc42 without reducing their expression. Consistently, the administration of Pl 8 to the mouse femora, inoculated with 4T1.2 mammary tumor cells, significantly suppressed the progression of tumor progression. Furthermore, molecular docking analysis predicted the binding of ARHGDIA and Pl 8 to Cdc42, and FRET-based live-cell imaging revealed that Pl 8 greatly reduced the activities of Cdc42 and RhoA.

[0378] Many chemotherapeutic medications, including DNA-toxic drugs and molecularly targeted antitumor therapies, exhibit myelosuppressive effects. Therefore, it is imperative to minimize anticancer drug doses while maintaining therapeutic efficacy. The combination of P18 with two widely used chemotherapeutic agents, Cisplatin and Taxol, effectively reduced ICso values. Subsequent application of human triple-negative breast cancer tissue fragments demonstrated that the concurrent administration of Pl 8 was more effective than using Cisplatin or Taxol alone. Moreover, co-culturing the mammary tumor cell line 4T1.2 with mouse femora revealed that P18 significantly down-regulated the generation of oncoproteins and RANKL while up-regulating alkaline phosphatase that is linked to bone formation. The result suggests that Pl 8 possesses osteoprotective properties in addition to its tumor-suppressing capabilities.

[0379] The iTSC-driven ACP candidates explored in this study set themselves apart from those in other ACP investigations due to their unique origin — peptide fragments derivedfrom tumor-suppressing proteins. Recent advancements in ACP development have focused on enhancing targeting and selectivity. Despite these strides, a majority of ACP clinical trials listed in the U.S. NIH Clinical Trials database are associated with peptide-based vaccines. Notably, vaccines such as p53 and K-RAS target the detection and elimination of malignant cells harboring mutations in p53 and K-Ras. While the side effect of any ACP is a concern, given that Pl 8 originates from proteins naturally produced by iTSCs, a potential lack of significant immune responses would be expected.

[0380] Pl 8 presents distinctive advantages and a comprehensive research profile. Despite the challenges associated with predicting effective ACPs, Pl 8 shows promise as a potent and efficient ACP. Notably, Pl 8 demonstrates a strong inhibitory effect on PDAC and OS cell lines. Furthermore, a thorough investigation into P18’s anticancer effects, including alanine substitution and two-terminal modifications, was conducted. The absence of hotspots for anticancer activity in alanine substitution suggests that the overall molecular structure of Pl 8 contributes to its anticancer properties. In acety lation and amidation experiments, Pl 8 modified with N-terminal acetylation and C-terminal amidation (AC-PI8-NH2) exhibited enhanced anticancer activity across multiple cancer cell lines. It is postulated that N-terminal amidation disrupts intramolecular interactions of P 18, thereby increasing its affinity to its target. Interestingly, P18S, a peptide composed of merely seven amino acids, demonstrated anticancer activity comparable to or surpassing that of Pl 8. The high industrial productivity and anticipated high bioavailability of short-chain peptides enhance the potential development of Pl 8S as an anticancer peptide. This outcome significantly bolsters the prospects for the advancement of anticancer peptide drugs derived from P18S.

[0381] ACPs are designed to bind specifically to tumor cells, facilitating direct tumor inhibition while minimizing adverse effects on normal tissues. This targeted approach positions ACPs as potent tools for individualized therapy, adaptable to the specificity of different patients’ tumors. The screening of iTSCs CM-derived ACPs with direct tumor-targeting capabilities holds immense promise in the realm of cancer therapy.

[0382] Conclusion.

[0383] This study demonstrated that P l 8 is a candidate for inhibiting breast cancer ACP that suppressed the viability, motility, proliferation, and invasion capability of the three breast cancer cell lines, as well as pancreatic cancer and osteosarcoma cell lines. Pl 8 can be applied with existing chemotherapeutic agents, and it reduces the IC50 of Cisplatin and Taxol. P18 inhibited the GTPase function of RhoA and Cdc42 without significantly affecting their expression.

[0384] Ethics Approval and Consent to Participate.

[0385] The experimental procedures using animals were approved by the Indiana University Animal Care and Use Committee (#SC330R, approved on 21 March 2021) and complied with the Guiding Principles in the Care and Use of Animals endorsed by the American Physiological Society. The usage of human breast cancer tissues was approved by the Indiana University Institutional Review Board (#1911155674, approved on 11 December 2019), and the tissues were received from Indiana University Simon Comprehensive Cancer Center Tissue Procurement Core.REFERENCES

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Claims

CLAIMS1. An anticancer peptide comprising at least 90% sequence identity with an amino acid sequence selected from any of SEQ ID NO: 1-23.

2. The anticancer peptide of claim 1, wherein the amino acid sequence is selected from SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5 or a sequence having at least 90% sequence identity thereto.

3. The anticancer peptide of claim 1, wherein the amino acid sequence comprises SEQ ID NO: 5 or a sequence having at least 90% sequence identity with SEQ ID NO: 5.

4. The anticancer peptide of claim 1, wherein the amino acid sequence comprises SEQ ID NO: 18 or SEQ ID NO: 23, or a sequence having at least 90% sequence identity7with SEQ ID NO: 18, or a sequence having at least 90% sequence identity with SEQ ID NO: 23.

5. The anticancer peptide of any of claims 1-4, wherein the sequence identity is at least 95%.

6. The anticancer peptide of any of claims 1-4, wherein the sequence identity is at least 99%.

7. The anticancer peptide of any of claims 1-4, wherein the sequence identity is 100%.

8. The anticancer peptide of any of claims 1-7, wherein one or more amino acids of the amino acid sequence is phosphorylated, glycosylated, nitrosylated, biotinylated, methylated, acetylated, or lipidated.

9. The anticancer peptide of claim 8, wherein one or more amino acids of the amino acid sequence is glycosylated and the one or more glycosylated amino acids are N-glycosylated, O- glycosylated, C-glycosylated, glypiated, or phosphoglycosylated.

10. A pharmaceutical composition comprising one or more anticancer peptides of any of claims 1-9 suspended in solution.

11. The pharmaceutical composition of claim 10, further comprising a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, pharmaceutically acceptable preservative, pharmaceutically acceptable antioxidant, pharmaceutically acceptable salt, pharmaceutically acceptable pH modulator, pharmaceutically acceptable buffer, or any combination thereof.

12. The pharmaceutical composition of either of claims 10 and 11, wherein the anticancer peptide comprises at least 99% purity.

13. The pharmaceutical composition of either of claims 10 and 11, wherein the composition is essentially free of peptides, nucleic acids, and cell fragments, other than the anticancer peptide.

14. A kit comprising: the pharmaceutical composition of any of claims 10-13; a container; a label; and instructions that provide methods of administering the pharmaceutical composition.

15. A method of generating anticancer peptides, the method comprising: contacting mammalian cells with one or more cell growth signaling pathway activators; growing the mammalian cells in culture medium; removing the mammalian cells from the culture medium; adding a protein digestion enzyme to the culture medium; and purilying peptides of about 100 amino acids or less in length from the culture medium.

16. The method of claim 15, wherein the mammalian cells are mesenchymal stem cells (MSCs).

17. The method of claim 15 or claim 16. wherein contacting mammalian cells with one or more cell grow th signaling pathway activators comprises contacting mammalian cells withtwo or more, three or more, four or more, or five or more cell growth signaling pathway activators.

18. The method of any of claims 15-17, wherein the cell growth signaling pathway activators are selected from Wnt signaling pathway activators, PI3K signaling pathway activators, FGF signaling pathway activators, and Notch signaling pathway activators.

19. The method of any of claims 15-18, wherein the cell growth signaling pathway activators are selected from cMyc, Oct4, Klf4, and Sox2, or variants, homologs, derivatives, or fragments of any thereof.

20. The method of any of claims 15-19, wherein the cell growth signaling pathway activators are selected from cMyc, Oct4, Klf4, and Sox2.

21. The method of any of claims 15-20, wherein the protein digestion enzyme is trypsin.

22. The method of any of claims 15-21, wherein removing the mammalian cells from the culture medium comprises one or more rounds of centrifugation.

23. The method of any of claims 15-22. further comprising filtering the culture medium.

24. The method of any of claims 15-23, wherein the culture medium comprises fetal bovine serum.

25. The method of any of claims 15-24, wherein the culture medium further comprises one or more antibiotics.

26. The method of any of claims 15-25, wherein the growing mammalian cells in culture medium is performed at about 37°C.

27. The method of any of claims 15-26, wherein the growing mammalian cells in culture medium is performed at about 5% CO2 atmosphere.

28. The method of any of claims 23-27, wherein the filtering is with a 0.22-pm polyethersulfone membrane.

29. The method of any of claims 22-28, wherein the centrifugation is at from about 2,000 to about 4,000 rpm.

30. The method of any of claims 15-29. wherein the purifying comprises extracting via ion exchange chromatography, gel permeation chromatography, medium-pressure reversed phase chromatography, or high-pressure reversed phase chromatography.

31. The method of any of claims 15-30. further comprising eluting the purified peptides.

32. A method of treating a cancer in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of the anti cancer peptide of any of claims 1-9; or administering to the subject a therapeutically effective amount of the pharmaceutical composition of any of claims 10-13; or administering to the subject a therapeutically effective amount of the composition of any of claims 46-49.

33. The method of claim 32, wherein the subject is a human.

34. The method of claim 32 or claim 33, wherein the cancer is osteosarcoma.

35. The method of claim 32 or claim 33, wherein the cancer is breast cancer.

36. The method of any of claims 32-35, wherein the cancer is metastatic.

37. The method of any of claims 32-36, wherein treating the cancer reduces a tumor size.

38. The method of any of claims 34-37. wherein treating the cancer increases bone volume ratio, increases bone mineral density, increases trabecular number, reduces trabecular separation, or reduces osteoclastogenesis, or a combination thereof.

39. A method of killing cancer cells, the method comprising: contacting the cancer cells with the anticancer peptide of any of claims 1-9.

40. The method of claim 39, wherein the cancer cells are human cancer cells.

41. The method of claim 39 or claim 40, wherein the cancer cells are osteosarcoma cells.

42. The method of claim 39 or claim 40. wherein the cancer cells are breast cancer cells.

43. Use of an anticancer peptide of any of claims 1-9; or a pharmaceutical composition of any of claims 10-13; or a composition of any of claims 46-49 for the manufacture of a medicament for the therapeutic treatment of cancer.

44. The use of claim 43, wherein the cancer is osteosarcoma.

45. The use of claim 43, wherein the cancer is breast cancer.

46. A composition comprising a polynucleotide encoding an anticancer peptide of any of claims 1-7.

47. The composition of claim 46, wherein the polynucleotide is encoded in a vector.

48. The composition of claim 47, wherein the vector is selected from the group consisting of plasmids, viral vectors, bacteriophages, cosmids, artificial chromosomes, and any combination thereof.

49. The composition of claim 48, wherein the viral vector comprises an adeno-associated virus.

50. A kit comprising: the composition of any of claims 46-49; a container; a label; and instructions that provide methods of administering the composition.

51. The method of claim 32 or claim 33, wherein the cancer is any of osteosarcoma, breast cancer, or pancreatic cancer.

52. The method of claim 32 or claim 33, wherein the cancer is pancreatic cancer.

53. The method of claim 52, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

54. The method of any of claims 32, 33, or 51-53, wherein the cancer is incipient.

55. The method of any of claims 51-54, wherein treating pancreatic cancer treats canceronset diabetes or cancer-related jaundice or both.

56. The method of claim 39 or claim 40, wherein the cancer cells are pancreatic cancer cells.

57. The method of claim 55 wherein the pancreatic cancer cells are pancreatic ductal adenocarcinoma cells.

58. The use of claim 43 wherein the cancer is pancreatic cancer.

59. The use of claim 58 wherein the cancer is pancreatic ductal adenocarcinoma.

60. A kit comprising: the anticancer peptide of any of claims 1-9; a container; a label; and instructions that provide methods of administering the anticancer peptide.

61. The anticancer peptide of claim 1, wherein the amino acid sequence comprises SEQ ID NO: 4 or a sequence having at least 90% sequence identity with SEQ ID NO: 4.

62. The anticancer peptide of claim 61, wherein the sequence identity is at least 95%.

63. The anticancer peptide of claim 61, wherein the sequence identity is at least 99%.

64. The anticancer peptide of claim 61, wherein the sequence identity is 100%.

65. An anticancer peptide, a pharmaceutical composition, a kit, a method, or a use as substantially described herein.

66. An anti cancer peptide comprising at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 18 or SEQ ID NO: 23.

67. An anticancer pharmaceutical composition comprising the anticancer peptide of claim 66.

68. The anticancer peptide of claim 66, wherein the peptide comprises an N-terminus acetyl modification, a C-terminus amino-group modification, or both an N-terminus acetyl modification and a C-terminus amino-group modification.

69. The anticancer pharmaceutical composition of claim 67, further comprising cisplatin, taxol, or both cisplatin and taxol.

70. An anticancer pharmaceutical composition comprising the anticancer peptide of claim 68.

71. The anticancer pharmaceutical composition of claim 70, further comprising cisplatin, taxol, or both cisplatin and taxol.

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