Methods for modifying macrophage differentiation and immunity

Abstract

To provide macrophages or monocytes for tumor treatment. [Solution] A method for treating cancer, comprising preparing genetically modified macrophages or monocytes containing a nucleic acid sequence encoding a Hom-1 polypeptide or a fragment thereof comprising the Hom-1 homeobox domain, wherein the nucleic acid sequence is operably linked to a heterologous promoter so that the modified macrophages or monocytes express the Hom-1 polypeptide or fragment thereof, and administering the modified macrophages or monocytes to a subject with cancer.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application is a continuation of U.S. Provisional Patent Application No. 62 / 253,8 filed November 11, 2015. No. 36, the entire contents of which are incorporated herein by reference. It forms part of a book. [Background technology]

[0002] The role of immunity in carcinogenesis is becoming better understood. Macrophages play an important role in innate immunity. are recognized as key components of tumors and the tumor microenvironment, acting as agents of both adaptive and adaptive immunity. Extensive research has demonstrated that tumor-associated macrophages play a key role in the growth, invasion, and metastasis of almost all tumors. The role of TAMs, which are derived from circulating monocytes, was suggested. A broad spectrum ranging from an M1-like phenotype in early stage tumors to an M2-like phenotype in most advanced tumors Consistent with their role in promoting tumorigenesis, M2-like TAMs display a phenotype similar to that of I Increased expression of L-10, IL4, MMP, and VEGF, which are pro-inflammatory cytokines , and reduced expression of cytotoxic iNOs and ROI. In addition to its intrinsic function in promoting tumorigenesis, TA M also plays a role in alternating T cell responses and balance in the tumor microenvironment. This contributes to the suppression of antitumor immunity. . Summary of the Invention [Problem to be solved by the invention]

[0003] However, the efficacy and potential applications of targeted TAMs in tumor therapy remain unclear. However, there is limited understanding of how TAM plasticity is regulated by intracellular factors. It was restricted because of that. [Means for solving the problem]

[0004] In one aspect, provided herein is a method for treating cancer, the method comprising administering to a subject an antibody against Hom-1 A polypeptide or a fragment thereof comprising the Hom-1 homeobox domain. providing a genetically modified macrophage or monocyte comprising a nucleic acid sequence comprising wherein the nucleic acid sequence is operably linked to a heterologous promoter and The present invention relates to a method for treating cancer by the expression of Hom-1 polypeptide or a fragment thereof in lymphocytes or monocytes. and administering the modified macrophages or monocytes to a subject. The modified macrophages or monocytes may be macrophages or monocytes derived from the subject or from another subject. The modified vector is produced by introducing an exogenous expression construct into a host or monocyte. The macrophages exhibit an M1 phenotype. and, in a subject, lower levels of Hom-1 expression in tumor-associated macrophages. It may further include detecting.

[0005] In another aspect, a method of treating cancer comprises administering to a macrophage or monocyte a gene encoding Hom-1 contacting the macrophages or The expression level of endogenous Hom-1 in monocytes was higher than that before the contact step. and administering the macrophages or monocytes thus contacted to a subject having cancer. Including, methods are described herein.

[0006] Also provided is a method for treating cancer, comprising inducing expression of the M1 gene in macrophages or monocytes. or an agent that inhibits expression of the M2 gene, The present invention relates to the generation of macrophages exhibiting the M1 phenotype, and the administration of the macrophages thus generated. and administering the phage to a subject having cancer.

[0007] In yet another aspect, described herein is a method of identifying cancer in a subject. The study identified genes involved in macrophages derived from the microenvironment of tissue areas suspected to be cancer. detecting the expression level of a gene selected from the group consisting of Hom-1 gene, M1 gene, and M 2 genes, where (i) lower levels of H (ii) a higher expression level of the M2 gene; Detection indicates that the suspicious tissue area is cancerous or at risk for developing cancer.

[0008] Also described in this disclosure is a method for identifying a candidate compound for the treatment of cancer, the method comprising: contacting a test cell with a test compound, wherein the test cell is a macrophage; or monocytes, and in the test cells, (i) Hom-1, (ii) Hom-1 pro (iii) a reporter gene operably linked to a motor; (iv) an M1 gene; or ) Detecting the expression level of the M2 gene and comparing the expression level with the corresponding control level. and selecting a test compound that modulates the IL-1 domain, wherein the selected compound is a compound that is capable of treating cancer. In one embodiment, the method comprises: contacting the phage with the selected compound; and and assaying the phage for anti-tumor activity. The advanced macrophages may be tumor-associated macrophages or M2 macrophages. The process can be carried out with the test cells in co-culture with the cancer sample. M1 macrophages, M2 macrophages, tumor-associated macrophages, tissue macrophages The cell may be selected from:

[0009] In one aspect, a method for identifying a candidate compound for the treatment of cancer comprises: and providing a co-culture comprising the test cells being macrophages or monocytes. and adding a test compound to the co-culture, and (i) inhibiting the cancer sample, as compared to a control. (ii) operably linking Hom-1 or the Hom-1 promoter in the test cell; (iii) increasing the expression level of a linked reporter gene in the test cell; (iv) increasing the expression of the M2 gene in the test cells. or (v) operably coupled to Hom-1 or the Hom-1 promoter in the test cell. A test compound that inhibits a significant decrease in the expression level of the linked reporter gene is selected. and b) determining whether the selected compound is a candidate compound for the treatment of cancer. In one embodiment, the cancer sample is a cancer tissue sample. In the method, the test cells and the cancer sample can be in direct or indirect contact with each other. The cells are classified into M1 macrophages, M2 macrophages, tumor-associated macrophages, and tissue macrophages. The antibody may be selected from a phage, and a monocyte-derived macrophage.

[0010] In another aspect, a method for identifying a candidate compound for the treatment of cancer comprises: contacting the tissue sample with a test compound; and determining whether (i) the tissue is (ii) inhibiting H in tumor-associated macrophages or monocytes in the tissue sample; (iii) increasing the expression level of om-1 in tumor-associated macrophages in tissue samples; (iv) increasing expression of the M1 gene in tumor-associated macrophages or monocytes; (v) reducing the expression of M2 genes in phage or monocytes; or (v) in a tissue sample. The expression level of Hom-1 in tumor-associated macrophages or monocytes was significantly decreased in and selecting a test compound that inhibits the expression of the agonist, wherein the selected compound is useful for treating cancer. The methods described herein are directed to determining whether a compound is a candidate for the

[0011] The details of one or more embodiments are set forth in the detailed description below. Other features of the embodiments include: Objects and advantages will become apparent from the detailed description and claims. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Surprisingly, Hom- in TAMs was significantly higher than in macrophages isolated from normal tissues. Surprisingly, Hom-1 expression in TAMs was significantly decreased. Increased expression of IL-1 transformed TAMs into M1-like macrophages with tumoricidal activity. It was further discovered that

[0013] The human homeobox transcription factor Hom-1 acts as an antagonist of canonical Wnt signaling. The nucleic acid sequence of Hom-1 (SEQ ID NO:1) and the amino acid sequence encoded by Hom-1 are shown in Table 1. The nucleotide sequence (SEQ ID NO: 2) is shown below: acctggccgc c atgcgcctc tcctcctccc cacctcgtgg cccgcagcag ctctccagct ttggctccgt gga ctggctc tcccagagca gctgctcagg gccgacccac acccccaggc ctgccgactt ctccctgggg agcctc cctg gcccaggcca gacatccggc gcccgggagc cccctcaggc cgtcagcatc aaggaggccg cgggtcct c aaatctgcct gcgccggaga ggaccatggc cgggttgagt aaggagccaa ataccttgcg ggccccccgt g tccgcacag ccttcaccat ggagcaggtc cgcaccttgg agggcgtctt ccagcaccac cagtacctga gccc tctgga gcggaagagg ctggccaggg agatgcagct ctcagaggtc cagataaaaa cctggttca gaatcgc cgc atgaaacaca aacggcaaat gcaggacccc cagctgcaca gccccttctc ggggtctc catgcgcccc cagctttcta ctcaacgtct tctggccttg caatggcct gcagctgctg tgcccttggg caccctgtc cg ggccccag gctctgatgc tgccccctgg ctccttctgg ggtctctgcc aagtggcaca agaggccctg gcatc tgcgg gagcttcctg ctgcgggcag cctctggcgt cccacccccc taccccaggc cggccttcgc tgggacca gc cctgtccacg gggccccggg gcctgtgtgc tatgccacag acgggggatg cattttga gg aggcacctct gactcccaca ctcgcggtct tgctgatcgc acctggctcc tacctggagg actcagttgt tctgtttaca tcc tggtggc acctctcacc ctgacccaca caaaggttct ggagattact ggagaatata tataaatata tatatg tacg tatatatgta aatacacata tacgtatata taaatatata tatacatatg tgtgtgtata tatatatat a tttttttttt tttttttttt tttgagacgg agtgttgctc tgtcacccag gctggagtgc aatgacgcaa t ctcggctca ctgcaacctc cgcctcctgg gttcaagcga ttctccagcc tcagcctccc gagtagctgg gatt acagac acccgccacc acgcccggct aattttttct atttttagta gaaatggggt ttcaccatgt tagccag gct ggtctcaaac tcctgaccct gtgatccgcc cgcctcggcc tcccaaagtg ctgggattac aggcatgagc cactgcaccc ggccctgaga atatatttat taaagccacc tcttcactga aagttaccga aagagtcggt tt aggaagga aacgaagggt cagtgaacag agtcaaatgc agaagtgggc ttgtcatggg tagggctttc ggcgt acgat aaaaggatca tttgtttttt aaaggggtt ggaaaaactg gttttccagt tggaaacagt aaaggttg ta agctttgtgt gtacaaaaga aaacagggaa tgcaggtgtg tttatagcgt tgtggttcaa gtccctctta acaagaactc caaagctgga aagcaggagg gaacaaaggt gaacatgaag gcgaggatgc tggggccctg cag tgcgctc taggctgtgc gtgagccggg actgtaccca cagcttgctg agggctgctc ttcttgggcc agggaa agca gggcagccgg gacctgcggc tgtgcctgga ctgaagctgt cccgcaggtc cccaccctcc aacacgtgc t cacctgtccc cctcctcgca gcagcctcgg gacaaaacaa tgactcaagg acagcacttc tcgcagaagg t ctggaagtg cccagaatgg gaggcacgga agcccctccc ggggaggact ccccgttga tggaccgttc ttgg tgcaga ctcctgactg cgtgcatgaa acctgagaca agtgcaattc cttccatgtc gccccagagt gcccagg agg caggcagtgc ggggtgccca ggcagacggg ttcagcctgc agaactggag gcgacctgtg aaacccaccc gggcacccca acaggaacag aagcgtggtc ctgcggctgc gtccccagcg agtttcactt tccccttgct cg tttctccc ttgttgtaag tgtttacaac tggcatgtgc ttttaaacgt caggtaagag gggaacagct gctgt acatc gtcctggcga gtgacaatgt gacagaagcc tgggcgaggc cctcggaggg cagcagctgg acaggggc ta ctgggtttgg cctggacagc actgatttgt ggatgtggat gggggcacgt tgtccgtgat aaaagtacaa gtgcccctca caaaaaaaaa aaaaaaa (SEQ ID NO: 1, underlined: coding sequence) mrlssspprg pqqlssfgsv dwlsqsscsg pthtprpadf slgslpgpgq tsgareppqa vsikeaagss nlp apertma glskepntlr aprvrtaftm eqvrtlegwf qhhqylsple rkrlaremql sevqiktwfq nrrmx krqm q dpqlhspfs gslhappafy stssglangl qllcpwapls gpqalmlppg sfwglcqvaq ealasagas c cgqplashpp tpgrpslgpa lstgprglca mpqtgdaf (SEQ ID NO: 2, underlined: homeodomain)

[0014] The present invention relates to a method for treating cancer in a subject by administering to the subject macrophages that exhibit anti-tumor activity. As used herein, unless otherwise specified, "anti-tumor "Macrophages exhibiting M1 phenotype," "M1-like macrophages," and "Macrophages exhibiting M1 phenotype" The terms "prophages" and "prophages" can be used interchangeably.

[0015] M1-like macrophages are characterized by: (1) expression of Hom-1 in macrophages or monocytes; (2) increase the expression of one or more M1 genes in macrophages or monocytes. and / or (3) increasing the expression of one or more M2 genes in macrophages or monocytes. It can be produced by inhibiting expression.

[0016] Hom-1 expression was shown to be necessary and sufficient for differentiation of monocytes into macrophages. Therefore, monocytes (e.g., from the peripheral blood of a subject) can be used in the above method. In other words, increasing Hom-1 expression in monocytes can Can be differentiated into macrophages.

[0017] Macrophages or monocytes were cultured to express higher levels of endogenous Hom-1. It can be induced in vivo by various agents or treatments, such as LPS, cholera toxin (C TX), chemotherapy, radiation, cytokines (e.g., GM-CSF), phorbol 12 -myristate 13 acetate (PMA) and antibodies against inhibitors of Hom-1 expression can use RNAi to induce Hom-1 expression.

[0018] In addition, macrophages or monoclonal antibodies genetically engineered to express high levels of Hom-1 have been The spheres can be used to treat subjects with cancer. The phage or monocyte expresses the Hom-1 polypeptide or the Hom-1 homeobox domain. The nucleic acid sequence may include a nucleic acid sequence encoding a promoter or a fragment thereof. The modified macrophage or monocyte is operably linked to a Hom-1 polypeptide. Such modified macrophages or (macrophages) express the The modified monocytes (which can differentiate into anti- lymphocytes) express significantly higher levels of Hom-1 and exhibits tumor activity and / or M1 phenotype.

[0019] In addition, by introducing an additional copy of the Hom-1 gene into macrophages or monocytes, For example, endogenous macrophages or monocytes can be generated by A Hom-1 nucleic acid sequence operably linked to a Hom-1 promoter (Hom-1 A polypeptide encoding a polypeptide or a fragment thereof that contains the Hom-1 homeobox domain. An expression construct that inhibits the expression of a marker gene (eg, a marker gene) can be introduced into macrophages or monocytes.

[0020] Also, the Hom-1 polypeptide or a fragment thereof containing the Hom-1 homeobox domain is The fragments can be introduced into macrophages or monocytes by direct peptide delivery. .

[0021] Macrophages and monocytes are genetically transfected using methods known in the art. For example, an exogenous expression construct for expressing Hom-1 can be used. The antibody can be introduced (e.g., stably or transiently transfected) into macrophages or monocytes. In one embodiment, the Hom-1 nucleic acid sequence may be heterologous (i.e., Hom- operably linked to a constitutive or inducible promoter (not including the promoter of In one embodiment, the Hom-1 nucleic acid sequence is operably linked to an endogenous promoter. It is tied.

[0022] M1-like cells can also be produced by inducing expression of M1 genes in macrophages or monocytes. Can generate tumoricidal macrophages. Drugs that can induce M1 genes The agents include, but are not limited to, LPS, CTX, PMA, GM-SCF, INFγ, and chemokines. Macrophages or monocytes express high levels of the M1 gene. The M1 gene may be modified to induce IL1b, IL6, IL12, IL23, TNFα, iNOs, CD40, CD80, CD86, CD68, TLR4, TLR2, IL-1R, MHCII, CCL15, CCL20, CXCL9, CXCL1 , and SOCS3.

[0023] In addition, inhibition of M2 gene expression in macrophages or monocytes also inhibits M1-like It is possible to produce tumoricidal macrophages. IL4 agents (e.g., antibodies or RNAi agents), anti-IL13 agents (e.g., antibodies or RNAi agents) ), antibodies against the M2 protein, and RNAi agents targeting the M2 gene. M2 genes include ARG1, MMP9, CCL18, VEGF, IL10, IL4, TGFb, CD163, CD206, CD68, TLR8, TLR1, MHCII, TG M2, DcoyR, IL-1RII, Ym1 / 2, MMR / CD206, and SR. can be done.

[0024] Xenogeneic or autologous macrophages or monocytes were used to generate M1-like macrophages. When using heterologous macrophages or monocytes, HLA matching can be used. In addition, HLA-matched immunoglobulins can be used to avoid or minimize host reactions. Alternatively, macrophages or monocytes that are not resistant to the inflammatory process may be used. The method can be used to obtain autologous macrophages or monocytes from cancer patients.

[0025] The generated M1-like macrophages are administered by infusion or injection (e.g., intravenously, intrathecally, intramuscularly, , intracavitary, intratracheal, intraperitoneal, intracranial, subcutaneous, or via another type of intra-tissue route), percutaneous The pharmaceutical composition may be administered to a subject by direct administration, or by other routes known in the art. In one example, macrophages or monocytes are introduced into a site or tissue where a tumor is found. The antibody may be injected directly into tissues (eg, the liver or pancreas) or surrounding areas.

[0026] As much as needed (e.g., once a day to once every 30 days) and as often as needed (e.g., once a day to once a week) to treat cancer. The subject can be treated with M1-like macrophages for one to 30 times. The M1-like macrophages described in the document were treated with radiation, chemotherapy, antibodies, small molecule drugs, etc. The present invention can be used in combination therapy with other cancer therapies.

[0027] The data presented below demonstrate that Hom-1 expression mediates TAM killing, regardless of tumor type. Therefore, any cancer, especially those with low levels of Hom-1, may be transformed into tumor cells. Cancers associated with TAMs expressing M1-like macrophages as described herein Prior to treating a subject with the M1-like macrophages described above, The expression levels of Hom-1 in TAMs in subjects were compared to the expression levels seen in controls ( (e.g., macrophages found in normal tissues of the subject) may be useful.

[0028] Examples of cancers that can be treated by M1-like macrophages include, but are not limited to: Leukemia, sarcoma, osteosarcoma, lymphoma, melanoma, glioma, glioblastoma, pheochromocytoma, liver cancer, oocyte tumor Cancer of the bladder, skin cancer, testicular cancer, stomach cancer, pancreatic cancer, kidney cancer, breast cancer, prostate cancer, colorectal cancer, head and neck cancer, Brain tumor, esophageal cancer, bladder cancer, adrenocortical cancer, lung cancer, bronchial cancer, thyroid cancer, endometrial cancer, nasopharyngeal cancer carcinomas and sarcomas such as cancer, cervical cancer, liver cancer, metastatic cancer, and cancer of unknown primary site. .

[0029] Compared to the expression level of a control (e.g., the corresponding level in macrophages in normal tissues). In comparison, lower expression levels in macrophages found in the tissue microenvironment Detection of the Hom-1 or M1 genes in the cells, or the higher expression levels of the M2 gene, This indicates that the tissue area is cancerous or at risk of developing cancer. Methods for identifying whether a tissue region is cancerous or at risk for developing cancer are also provided herein. It is contemplated in the specification.

[0030] Further described herein are methods for identifying candidate compounds for the treatment of cancer. That is, cells (e.g., macrophages or monocytes) can be contacted with a test compound, and the test cells (i) Hom-1; (ii) a reporter gene operably linked to the Hom-1 promoter; (iii) an M1 gene; and (iv) an M1 promoter operably linked to the M1 gene. (v) a reporter gene operably linked to the M2 gene, or (vi) an M2 promoter. The expression level of the reporter gene linked to the control is detected. (i) to (iv) expression, as compared to the corresponding level in test cells not containing the Tests to increase the level and / or decrease the expression level of (v) or (iv) The compounds are candidate compounds for the treatment of cancer.

[0031] In one screening method, a test compound is added to a co-culture medium comprising a test cell and a cancer sample. The test compound is added to the test cells to determine whether it inhibits (i) Hom-1, A reporter gene operably linked to the Hom-1 promoter, the M1 gene, or increases the expression level of a reporter gene operably linked to the M1 promoter (ii) in the test cell, Hom-1, operably linked to the Hom-1 promoter, operably linked to a reporter gene, M1 gene, or M1 promoter (iii) inhibits a significant decrease in the expression level of the reporter gene in the test cell; and the M2 gene or a reporter gene operably linked to the M2 promoter. If the expression level is decreased, the test compound is a candidate compound for the treatment of cancer.

[0032] In the co-culture system, it is determined whether the test compound has an inhibitory effect on the cancer sample. It is also possible to inhibit the cancer sample (e.g., inhibit the growth of cancer cells, The test compound is selected from the group consisting of compounds that kill cancer cells or reduce the size of cancer specimens, and are useful in treating cancer. The test cells and the cancer sample may be directly contacted with each other. Alternatively, The test cells and the cancer sample can be mixed (e.g., by using transwell inserts). The cancer sample is a sample containing cancer cells, e.g., a cancer tissue sample, The cancer tissue sample may be an isolated cancer cell or a cell of a cancer cell line. Such cancer tissue samples may contain TAMs. do.

[0033] Also, the screening method can be performed using a cancer tissue sample in the absence of test cells. The cancer tissue sample may be contacted with a test compound. The TAMs may then be isolated from the cancer tissue sample. To identify the expression levels of Hom-1, M1, or M2 genes in TAMs. Alternatively or additionally, the expression level of Hom-1 in the tissue sample can be determined. The test compound may be used to detect whether the test compound inhibits (i) Hom-1 or increases the expression level of the M1 gene, (ii) the expression of Hom-1 or M1 gene and / or (iii) inhibiting a significant decrease in the expression level of the M2 gene. If the compound inhibits the proliferation of the cancer tissue sample compared to a control, the compound is a candidate for the treatment of cancer. Test compounds (e.g., to reduce sample size) may also be used to identify candidate compounds for the treatment of cancer. It is considered that.

[0034] The test compounds to be screened (e.g., proteins, peptides, peptidomimetics, peptide The therapeutic agent (e.g., peptide, antibody, RNAi, small molecule, or other drug) is known in the art. can be obtained using the methods described herein.

[0035] In any of the methods described herein, the Hom-1, M1 gene, or M2 gene The expression level of a gene can be determined at either the mRNA level or the protein level. It is also possible to measure promoter activity. mRNA levels, protein Methods for measuring the levels and activity of promoters are well known in the art.

[0036] In any of the screening methods described above, the test cells are macrophages. Macrophages may be M1 macrophages, M2 macrophages, or monocytes. 2 Macrophages, tumor-associated macrophages, tissue macrophages, or monocyte-derived The test cells may be macrophages or monocytes.

[0037] The following specific examples are to be construed as merely illustrative, and not limiting of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is understood that those skilled in the art will be able to fully understand the present invention based on the description herein. It is believed that the disclosures herein may be used in the art. All publications cited herein are incorporated by reference in their entirety. This document is hereby incorporated by reference in its entirety. EXAMPLES

[0038] The present inventors have demonstrated that the functional polarization of human macrophages is The present inventors identify the role of the human homeobox protein Hom-1 in the regulation of mitosis. Hom-1 promotes and is required for M1 activation, but not M2 activation, of human macrophages We found that it is not required for expression of critical genes involved in mitosis.

[0039] Using primary TAMs isolated from cancer, we compared the TAMs isolated from normal control tissues with those from normal control tissues. We found that Hom-1 expression was significantly reduced in TAMs compared to phages. The present inventors found that the expression profile of Hom-1 in TAMs correlates with the TAM phenotype. Furthermore, ectopic expression of Hom-1 converted TAMs to an M1-like phenotype. Both in vitro and in vivo data demonstrated that Hom-1 induces tumor-killing effects in TAMs. Taken together, our studies demonstrated that Hom-1 mediates TAM activation. We demonstrated that Hom-1-expressing TAMs (M1-expressing TAMs) convert the IL-1-binding domain into tumor-killing TAMs. showed that the α-terminal β-lactamase (α-lactamase) exerted a strong inhibitory effect on the growth of various cancers, making it useful for the treatment of cancer. These results suggest a role for Hom-1-regulating TAM as a new modality in the treatment of glaucoma.

[0040] Hom-1 expression was decreased in TAMs In advanced tumors, TAMs display a tumor-promoting M2-like phenotype. Bronte and Murray (2015) , Nat Med 21, 117-119. The plasticity of TAMs is well understood and various Cytokines have been implicated in the polarization of TAMs towards the M2 phenotype. Noy ​​and Poll ard (2014), Immunity 41, 49-61. In comparison, The transcription mechanism remains largely unknown.

[0041] As described below, discarded surgical specimens from colon cancer resections were used to identify tumor TAMs were isolated from the tissue and macrophages were isolated from normal mucosa 15 cm away from the tumor site. As previously noted, FACS analysis was performed on normal control mucosa. Compared to macrophages, TAMs expressed significantly higher levels of CD68, CD163, and CD2 06 expressed cell surface markers associated with the M2 phenotype. Zhang et al. (2013), Eur J Cancer 49, 3320-3334. In macrophages, the non-discriminating macrophage marker CD3 We found no significant difference in the expression of 3.

[0042] To determine whether Hom-1 may play a role in TAMs, we Hom-1 expression in TAMs was quantified by qRT-PCR compared with control macrophages. We found that Hom-1 expression was significantly reduced in TAMs compared with its expression in TAMs. .

[0043] VentX regulated TAM plasticity and polarized TAMs towards the M1 phenotype Previous studies have shown that LPS can induce TAMs to exhibit an M1 phenotype. See Zhang et al. for further details on whether Hom-1 plays a role in TAM plasticity. To determine whether Hom-1 expression is related to LPS exposure, we investigated the expression of Hom-1 in TAMs exposed to LPS. We found that Hom-1 expression was significantly increased in TAMs after stimulation with LPS. In parallel with the high expression of Hom-1, and consistent with previous findings, we found that T LPS stimulation of AM resulted in increased secretion of proinflammatory cytokines and cytotoxic iNOs. Ta.

[0044] To determine whether Hom-1 plays a regulatory role in TAM plasticity, we investigated the effect of Hom-1 knockdown on TAM phenotype. Treatment of TAMs with Folino (MO) resulted in approximately 80% reduction in Hom-1 expression. Consistent with a role for Hom-1 as a key regulator of TAM plasticity, In this study, we demonstrated that Hom-1 MO suppresses inflammatory cytokines and cytotoxicity in TAMs. We found that CD206 blocks LPS-induced secretion of iNOs. It is an M2 cell surface marker that is highly expressed in the endothelium and TAMs. Reflecting this, exposure of TAMs to LPS resulted in a significant reduction in the CD68+CD206+ population and C Hom-1 MO increased the number of CD68+CD206- cells. Both of these effects of LPS were abolished (p<0.01).

[0045] The correlation between Hom-1 expression levels and TAM phenotypes suggests that Hom-1 regulates TAM plasticity. This prompted us to further explore the idea that TAMs may play a role in the pathogenesis of GPF-Hom The mice were transfected with plasmids encoding GFP-1 or control GFP. TAMs transfected with GFP-Hom-1 compared to TAMs transfected with GFP-Hom-1. showed characteristic M1 morphology with elongated / fibroblast-like cell shape. FACS analysis Surface expression of M1 markers CD40, CD80, and CD86 was increased by GFP-Hom The results showed that the expression of GFP-1 in TAMs was significantly increased. -Hom-1-transfected TAMs upregulated the proinflammatory cytokine TNF The secretion of M2 cytokines was significantly increased, whereas the secretion of IL-1β, IL-12, and IL-1α was significantly increased. Consistently, gene expression analysis showed that GFP-H IL-1β, IL-6, TNF-α, and The expression of M1 genes such as iNOs and iNOs was significantly increased in the transfected cells with GFP-Hom-1. In transfected TAMs, M2 genes such as CCL18, MMP9, VEGFA, and Arg1 were upregulated. Taken together, our data suggest that Hom-1 is a These results suggest that it regulated TAM plasticity and promoted M1 polarization of TAMs.

[0046] Hom-1 converted TAMs into tumor suppressor cells Our finding that Hom-1 promotes M1 polarization of TAMs is This prompted us to explore whether mutant TAMs could exert tumor suppression. Freshly isolated TAMs were transfected with plasmids encoding GPF-Hom-1 or a control GFP. The Transwell culture system was then used to transfect tumors or The engineered TAMs were co-cultured with normal tissues. Notably, after 7–10 days of co-culture, After incubation with GFP-Hom-1 modified TAMs, tumor volume was significantly reduced. The effect was significantly reduced (approximately 70%) in GFP-transfected TAMs or There was no significant change in size in tumors incubated with TAM alone. To determine whether the volume reduction was associated with a reduction in cancer cells, we performed histological sectioning. The results were analyzed by immunohistochemistry using H&E staining and CK20 antibody in colon cancer cells. The authors demonstrated that tumor nests incubated with TAM or GFP-modified TAM However, it was found that CK20-positive tumor cells appeared in the cord and sheet. However, CK20-positive tumor cells co-expressed with TAMs transfected with GFP-Hom-1. The expression of Hom-1 disappeared during incubation with either GFP or GFP-Hom-1. During incubation with the transfected TAMs, the Hom-1 modified TAMs The finding that there was minimal effect on the volume and morphology of normal colonic mucosa led to the conclusion that Hom The specificity of the tumoricidal effect of -1-modified TAMs was demonstrated.

[0047] Hom-1 promotes the tumoricidal function of TAMs in vivo Our findings that Hom-1 converted TAMs to tumor-killing cells in vitro Our findings highlight a possible role for Hom-1-regulated TAMs in tumorigenesis in vivo. The colon cancer was cut into approximately 0.5 cm pieces and placed on the ventral side of NSG mice. One week later, MO-Hom-1-transfected TAMs ( Hom-1 inhibition) or GFP-Hom-1 transfected TAMs (Hom-1 expression) , and injected into the tail vein of mice. Eight weeks after xenografting, MO-Hom-1 transfected mice were Mice injected with TAMs developed tumors, whereas GFP-Hom-1-transfected T No tumors developed in mice injected with AM.

[0048] The present inventors also cultured TAMs or monocytes in M1 differentiation medium. We found that these could be induced to exhibit an M1 phenotype. TAMs / monocytes could be injected into NSG mice and inhibit cancer growth in vivo. The effect of M1-differentiated TAMs on tumor growth was due to the association of these TAMs or Hom It is abolished by inhibition of -1 expression.

[0049] Effect of ectopic expression of Hom-1 in TAMs on various types of cancer TAMs have been implicated in the oncogenesis of essentially all tumors. Following our work on AM, we extended our investigations to other types of tumors.

[0050] Surgical specimens of lung cancer, melanoma, esophageal cancer, gastric cancer, and pancreatic cancer were obtained and treated with T as described above. AMs were isolated. Macrophages were obtained from matched normal tissues of the same patients. TAMs and tissue Hom-1 expression in tissue macrophages was quantified using real-time RT-PCR. Hom-1 expression in TAMs from all these tumors was significantly higher than that in TAMs from distant normal tissues. Expression of Hom-1 in macrophages was low compared with that in the corresponding macrophages.

[0051] To determine whether Hom-1 could convert these TAMs into tumor-killing cells To investigate this, GFP or GFP-Hom-1 was transfected into TAMs. 48 hours after injection, GFP-positive cells were sorted and co-cultured with individual tumors. Tumor volume was reduced during coculture with GFP-Hom-1-transfected TAMs. However, there was no decrease when co-cultured with control GFP-transfected TAMs. Our results demonstrate that Hom-1 converts TAMs into tumor-killing cells, regardless of tumor type. This suggests that it was possible.

[0052] Collection of colon tissue samples In the pathology laboratory, cancer tissue and normal tissue are extracted from surgically resected specimens from patients. Approximately 5 to 10 grams of tissue was collected from each tumor or normal tissue 15 cm away from the tumor. Patients were also given blood samples.

[0053] Preparation of intraepithelial lymphocytes We modified a previously described technique (Kamada N, et al, 2008; Pignata C, et al, 1990). Briefly, freshly dissected mucosa and Tumors were cultured in 2% fetal bovine serum (FBS) and 1 mM dithiothreitol (DTT) (Sigma-Aldrich). Aldrich), Ca 2+ Free and Mg 2+ Free Hanks Balanced Salt Solution (HBS The mucus was removed by rinsing in a 10 cm Petri dish with Life Technologies. The tumors were cut into 0.5 cm pieces using a razor blade and then resuspended in 1 mM EDTA (Sigma-Aldrich Incubate in a 6-well plate with 5 mL of HBSS containing After filtering, the mixture was passed through a gray mesh (100 microns). This included intraepithelial lymphocytes and epithelial cells, and was analyzed by flow cytometry.

[0054] Isolation of macrophages from tumor masses and normal mucosa Then, 2% FBS, 1.5 mg / mL collagenase D (Roche), 0.1 mg / mL HBSS containing Dnase I (Ca 2+ and Mg 2+ (including The mucosa and tumor were incubated for 24 h. The digested tissue was passed through a filter. The flow-through was collected and purified with 40% Percoll solution (Pharmacia ), then layered on 60% Percoll and run at 2000 rpm without interruption (with The cells were centrifuged for 30 minutes (out brake). The LPMCs at the interface were collected. According to the study, without depletion of CD16, EasySep™ human monocyte / macrophage concentrate was used. Normal mucosal macrophages were isolated from LPMCs using a cloning kit (StemCell Technologies). Cells isolated by these techniques were purified using propidium iodide (P I) Staining consistently showed >98% viability. Purity of intestinal macrophages was >95%. there were.

[0055] Preparation of macrophages from peripheral blood Peripheral blood mononuclear cells (PBMCs) derived from healthy adult donors were used at Brigham and Women's Hospital. BMCs were isolated by Ficoll density gradient centrifugation according to the manufacturer's instructions. Without depletion of D16, PBMs were isolated using the EasySep™ Human Monocyte Enrichment Kit. Human monocytes were purified from C. monocytes. Purified cells were incubated with 10 ng / mL M-CSF (PeproTech). After enrichment, monocytes were cultured in complete RPMI medium containing M-CSF. The cells were then cultured for 5 days and used in the co-culture system.

[0056] FACS analysis After immunolabeling the cells with fluorescent dye-conjugated antibodies, flow cytometry was used to characterize the TA Phenotypic analysis of M and other lymphocytes was performed using the following antibodies: PE-conjugated anti- CD3 (OKT3), anti-CD25 (BC96), anti-CD14 (61D3), anti-CD68 ( eBio Y182A), anti-CD163 (eBio GH161), anti-CD206, FI TC-conjugated anti-CD4 (RPA-T4), anti-CD33 (HIM3-4), APC-conjugated Antibody antibodies against CD8 (OKT8) and CD4 (OKT4) (eBioscience, Inc.) were used. Foxp3 was detected using a PE-conjugated antibody according to the protocol provided by the manufacturer. (236A / E7), which performed intracellular staining for IFN-γ, perforin, and granzyme B. Isotope control labeling was performed in parallel. Antibodies were labeled as recommended by the supplier. The labeled cells were analyzed using Cell-Quest software (BD Biosciences). Acquire on a FACScan flow cytometer and analyze by FlowJo software Results are expressed as the percentage of positive cells.

[0057] Organotypic co-culture of tumors and macrophages 12 Transwell inserts (pore size 0.4 μm, Costar, Corning) 10-well polystyrene tissue culture plates (Beuren, Franklin Lakes, NJ) The mucosa and masses were weighed and placed in 1x PBS buffer containing antibiotics. After washing with PBS, the tissue was cut into 0.5 cm pieces. Approximately 50 mg of tissue was placed in a 12-well transfection chamber. The cells were placed in the upper compartment of the well and filled with 0.5 mL of complete RPMI 1640 medium. Add it. 5×10 5 TAMs were cultured at a density of 500,000 cells / well in direct cell-tissue contact. The lower compartment was filled with 2 mL of PRMI complete medium without any additional stirring. The plates were incubated at 37°C and 5% CO2. 0.5 mL of culture medium was added to the cytokines. After 2 weeks of co-culture, the lower chamber was harvested for in vitro analysis and fresh medium was added every 3 days. Macrophages from each group were collected and total RNA was isolated using TRIzol reagent (Ambion). Tumors and normal mucosa were monitored twice weekly with calipers (length × width). 2 ) / 2 Tumor volume was calculated according to the formula: 3 megapixels on a Leica EZ4D stereomicroscope. Take a picture of the tissue using a CMOS camera or an iPhone digital camera. I took a picture.

[0058] Immunohistochemistry Tumor or normal tissues were incubated in formalin (Fisher Scientific Company, Kalamazoo, MI). ) and stained with CK20 (Dako, Carpinteria, CA, clone Ks 20.8, 1:50) and hematoxylin / eosin (H&E) staining were performed. Epitope Retrieval 2 was used for 20 minutes in the The Ymer Refine detection kit was used to stain the Leica Bone III plate. CK20 staining was performed on the platform using a Nikon Eclipse Ti fluorescence microscope. Microscopic analysis was performed using NIS Elements image processing software (Nikon Images were captured at an original magnification of 40x using a color camera that applies Brightness and contrast for representative images were adjusted equally between groups.

[0059] Hom-1 overexpression The cells were transfected with Lipofectamine 2000 (Life Technologies) according to the manufacturer's protocol. We then transfected GFP-Hom-1 into blood macrophages and TAMs. 48 hours after transfection, the cells were filtered through a 70 μm cell sorting filter. The GFP-positive cells were then cultured under sterile conditions using Baker Bio-Protect H After sorting, cells were sorted using a BD FACSAria II under 5% CO2. Cultured in MI 1640 complete medium.

[0060] Hom-1 knockdown Human Monocyte Nucleofector Kit (Maryland, MD) was used according to the manufacturer's instructions. Colonic TAMs or human primary monocytes were cultured using morpholinotransferase (Lonza, Walkersville, VA, USA) to generate morpholinotransferases. The cells were transfected with MO antisense oligonucleotides. 0 6 Cells were incubated with 2.5 nmol of Hom-1 MO oligonucleotide or standard control M Add 100 µl nucleofector solution containing either the oligonucleotide The cells were suspended and electroporated using a Nucleofector II device (Lonza). After 2 h, the cells were immediately removed from the device and placed in pre-warmed 100 mL of 1000 mM glutamine and 10% FBS. The cells were incubated overnight with 1 ml of Human Monocyte Nucleofector Medium. The cells were then resuspended in complete RPMI medium and treated with appropriate cytokines to induce macrophage proliferation. Both MO oligonucleotides were purchased from Gene Tools (Philippines, OR). I ordered it from Lomas.

[0061] Cytokine measurements E. coli LPS (Sigma) was assayed using an ELISA kit obtained from eBiosciences. a-IL-1 in the supernatant of Aldrich-treated blood macrophages or LPS-treated TAM The levels of IL-12p70, IL-10, TNF-α, and IL-12 were quantified. The procedure was carried out according to the instructions given by the person in charge.

[0062] qRT-PCR Total RNA was isolated using TRIzol reagent, and the RNA amount was analyzed by NanoDrop 2000 (Thermo Scientific). Script III first-strand synthesis system (Life Technologies) Equal amounts of RNA were used for first-strand cDNA synthesis. To amplify om-1 cDNA, we used Accu The Prime Taq DNA polymerase system (Life Technologies) was used. The PCR products were separated on a 2% agarose gel and stained with ethidium bromide. The inventors used a LightCycler (480 Real- Hom-1 was analyzed using SYBR Green on a Time PCR System (Roche). and other gene cDNA quantitative measurements. Then, the comparative Ct method (DDCT method) was used. was used to calculate the relative expression profile of mRNA.

[0063] Arginase activity and NO assay QuantiChrom Arginase Assay Kit (DARG-200; BioAssays Arginase activity in cell lysates was assessed by measuring urea production using the ELISA Kit (ELISA Kit) (ELISA Kit). The nitrite concentration in the culture supernatant was measured using the Griess Reagent Kit (Molecula r Probes).

[0064] statistical analysis Student's test was used for statistical analysis.

[0065] Other embodiments All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be used as an alternative serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the disclosed Each feature listed is merely an example of a generic series of equivalent or similar features. do.

[0066] From the above detailed description, those skilled in the art can easily discern the essential characteristics of the described embodiments. and can be adapted to a variety of uses and conditions without departing from its spirit and scope. Various changes and modifications of the above embodiment can be made to fit the Other embodiments are within the scope of the claims.

Claims

1. 1. A method for identifying cancer in a subject in vitro, comprising: detecting the expression level of the gene in macrophages derived from the microenvironment of the tissue area suspected to be cancer; the gene is the Hom-1 gene, wherein detection of a lower level of Hom-1 gene expression compared to a corresponding control level indicates that said suspect tissue area is cancerous or at risk of developing cancer.

2. The method of claim 1 , wherein the control level is the level of macrophages in a normal tissue.

3. 1. A method for identifying a candidate compound for the treatment of cancer, comprising: contacting a test cell with a test compound, wherein the test cell is a macrophage or a monocyte; detecting in the test cell an expression level of (i) Hom-1, or (ii) a reporter gene operably linked to a Hom-1 promoter; selecting a test compound that increases the expression level of Hom-1 compared to a matched control as a candidate compound for treating cancer; A method comprising:

4. contacting tumor-promoting macrophages with the selected compound; assaying said tumor-promoting macrophages so contacted for anti-tumor activity; The method of claim 3 further comprising:

5. The method of claim 4, wherein the tumor-promoting macrophage is a tumor-associated macrophage or an M2 macrophage.

6. The method of any one of claims 3 to 5, wherein the contacting step is performed with the test cells in co-culture with the cancer sample.

7. 1. A method for identifying a candidate compound for the treatment of cancer, comprising: providing a co-culture comprising a test cell and a cancer sample, the test cell being a macrophage or a monocyte; adding a test compound to the co-culture; selecting a test compound that (i) increases the expression level of Hom-1 or a reporter gene operably linked to a Hom-1 promoter in said test cell, or (ii) inhibits a significant decrease in the expression level of Hom-1 or a reporter gene operably linked to a Hom-1 promoter in said test cell, relative to a corresponding control; wherein the selected test compound is a candidate compound for the treatment of cancer.

8. The method of claim 6 or 7, wherein the cancer sample is a cancer tissue sample.

9. The method of any one of claims 6 to 8, wherein the test cell and the cancer sample are in direct or indirect contact with each other.

10. The method of any one of claims 3 to 9, wherein the test cell is an M1 macrophage, an M2 macrophage, a tumor-associated macrophage, a tissue macrophage, or a monocyte-derived macrophage.