Post-ablation modulation in radiotherapy

Abstract

The present invention provides a method and system for treating cancer in a subject, the method comprising providing an ablation dose of radiotherapy to a first region containing cancer, and then providing a subablation dose to a second region, wherein the subablation dose is administered from 1 hour to 4 days after the ablation dose.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 892,273, filed August 27, 2019, which is incorporated herein by reference in its entirety.

[0003] Statement on Federally Funded Research

[0004] This invention was completed with government support under licenses granted by the National Institutes of Health (NIH) under license numbers R01CA22686(CG), 1S10OD019961-01, and 1S10RR029545-01. The government holds certain rights to this invention. Background Technology

[0005] Cancer is a leading cause of death worldwide and is currently the second leading cause of death in the United States. Conventional treatment options for cancer include cytotoxic therapies such as radiation therapy (RT) and chemotherapy. Even with systemic therapy, local tumor control achieved through surgery or RT can fail due to eventual metastatic progression. Recently, immunotherapy has been proposed as a potential treatment option to inhibit metastatic progression, and it can be used alone or in combination with other therapies. Immunotherapy includes checkpoint inhibitors, tumor vaccines, and adoptive cell transfer. While immunotherapy has shown promise in treating certain solid tumors, such as melanoma and renal cell carcinoma, it has been less successful in other, more fibrotic tumors, such as pancreatic cancer. Even within cancer types, there are subsets of responders and non-responders, meaning that treatment is not ideal for many patients.

[0006] While radiotherapy (RT) can be used to treat cancer, its cytotoxic effects limit its effectiveness. For example, RT on tumors located in organs can severely damage the organ where the tumor is located. Metastatic tumors can be located far from the primary tumor, resulting in primary and metastatic tumors being located in more than one organ. Due to its cytotoxic effects, RT on more than one organ can lead to damage to more than one organ, at least in some cases.

[0007] In light of the above, there is a desire for improved methods and devices for treating cancer. Ideally, these methods and devices would inhibit the metastatic progression of cancer and allow for the treatment of tumors in vulnerable organs and tissue structures that could cause damage. Summary of the Invention

[0008] This method and device provide improved treatment for cancer using radiotherapy (RT). Tumors can be treated with an ablation dose of radiation, followed by subsequent subablation therapy at the same or different sites. Subsequent subablation therapy can be applied from approximately 1 hour to approximately 4 days after ablation to provide an adaptive immune response to antigens on cancer cells. Subsequent subablation therapy can be applied to many sites, such as the same site on the tumor receiving the ablation dose, another tumor, or a site prone to metastasis. In some embodiments, the time between ablation and subablation allows for structural alterations in the tumor microenvironment, including the tumor's vascular system. Because the subsequent treatment is subablation, it can be applied to many sites, even unidentified tumor sites, and can include, for example, systemic therapy. Radiation therapy can be administered in various ways, such as using a radiation therapy machine or brachytherapy and combinations thereof.

[0009] While treatment can be performed in various ways, in some implementations, the initial ablation radiotherapy generates a large number of antigens that are presented to the immune system. An adaptive immune response is generated to the antigens presented in the first treatment. Subsequent subablation therapy can influence the tumor microenvironment and increase the extent to which immune cells can enter the tumor. Subsequent subablation therapy can lead to increased tumor perfusion (e.g., tumor “rupture”) to alter the tumor microenvironment and may result in other changes to the tumor microenvironment that promote an immune response. Although treatment of tumors with subablation doses of RT is mentioned, tumor-vulnerable tissues can also be treated prophylactically with RT to suppress tumor growth in those tissues.

[0010] In some embodiments, this document describes a method for treating cancer in a subject, the method comprising delivering an ablation dose of radiotherapy to a first region containing cancer, followed by a subablation dose to a second region, wherein the subablation dose is administered after the ablation dose. In some embodiments, the subablation dose is administered at least 1 hour after the ablation dose. In some embodiments, the subablation dose is administered at least 1 day after the ablation dose. In some embodiments, the subablation dose is administered no more than 4 days after the ablation dose.

[0011] In some embodiments, this document discloses a method for treating cancer in a subject, the method comprising delivering an ablation dose of radiotherapy to a first region containing cancer, followed by a subablation dose to a second region, wherein the subablation dose is administered from 1 hour to 4 days after the ablation dose. In some embodiments, the cumulative amount of radiotherapy delivered to the second region throughout the treatment includes less than an ablation dose. In some embodiments, the cumulative amount of less than an ablation dose includes multiple subablation doses. In some embodiments, the first region includes a tumor region and optionally, the second region includes a tumor region. In some embodiments, the first region includes a region of a first tumor and the second region includes a region of a second tumor. In some embodiments, the first tumor includes a primary tumor and the second tumor includes a metastatic tumor. In some embodiments, the first tumor includes a metastatic tumor and the second tumor includes a primary tumor. In some embodiments, the second region includes multiple second regions and each of the multiple second regions receives a cumulative amount of radiotherapy less than an ablation dose. In some embodiments, the second region includes a region different from the first region. In some embodiments, the second region includes a tumor region. In some embodiments, the second region includes areas that may develop into metastatic tumors, and optionally, the second region includes areas of organs selected from the group consisting of: bone, lymph nodes, lungs, liver, brain, adrenal glands, breast, eyes, kidneys, muscles, pancreas, salivary glands, and spleen. In some embodiments, the second region includes the entire body of the subject scanned with a subablation dose. In some embodiments, the first region includes areas of primary tumors in organs selected from the group consisting of: breast, bladder, brain, colon, rectum, endometrium, kidneys, pancreas, prostate, liver, lungs, skin, thyroid gland, uterus, lymph nodes, tonsils, thymus, spleen, and bone marrow, and the second region includes areas of metastatic tumors in organs selected from the group consisting of: bone, lymph nodes, lungs, liver, brain, adrenal glands, breast, eyes, kidneys, muscles, pancreas, salivary glands, and spleen. In some embodiments, the first region includes a region of metastatic tumors in organs selected from the group consisting of: bone, lymph nodes, lung, liver, brain, adrenal glands, breast, eye, kidney, muscle, pancreas, salivary glands, and spleen, and the second region includes a region of primary tumors in organs selected from the group consisting of: breast, bladder, brain, colon, rectum, endometrium, kidney, pancreas, prostate, liver, lung, skin, thyroid gland, uterus, lymph nodes, tonsils, thymus, spleen, and bone marrow. In some embodiments, the first region includes identified tumors, and the second region does not include identified tumors.

[0012] In some embodiments, this document discloses a computer-readable medium configured with instructions that, when executed, cause a processor to instruct a radiotherapy system to deliver an ablation dose of radiotherapy to a first region, and then a subablation dose to a second region following the ablation dose. In some embodiments, the subablation dose is delivered at least 1 hour after the ablation dose. In some embodiments, the subablation dose is delivered at least 1 day after the ablation dose. In some embodiments, the subablation dose is delivered no more than 4 days after the ablation dose.

[0013] In some embodiments, this document discloses a computer-readable medium configured with instructions that, when executed, cause a processor to instruct a radiotherapy system to deliver an ablation dose of radiotherapy to a first region, and then to deliver a subablation dose to a second region within 1 hour to 4 days after the ablation dose. In some embodiments, the cumulative amount of radiotherapy delivered to the second region throughout the treatment includes an amount of radiotherapy less than the ablation dose. In some embodiments, the cumulative amount of radiotherapy less than the ablation dose includes multiple subablation doses. In some embodiments, the first region includes a tumor region, and optionally, the second region includes a tumor region. In some embodiments, the first region includes a region of a first tumor, and the second region includes a region of a second tumor. In some embodiments, the first tumor includes a primary tumor, and the second tumor includes a metastatic tumor. In some embodiments, the first tumor includes a metastatic tumor, and the second tumor includes a primary tumor. In some embodiments, the second region includes multiple second regions, and each of the multiple second regions receives a cumulative amount of radiotherapy less than the ablation dose. In some embodiments, the second region includes a region different from the first region. In some embodiments, the second region includes a tumor region. In some embodiments, the second region includes areas that may develop into metastatic tumors, and optionally, the second region includes areas of organs selected from the group consisting of: bone, lymph nodes, lungs, liver, brain, adrenal glands, breast, eyes, kidneys, muscles, pancreas, salivary glands, and spleen. In some embodiments, the second region includes the entire body of the subject scanned with a subablation dose. In some embodiments, the first region includes areas of primary tumors in organs selected from the group consisting of: breast, bladder, brain, colon, rectum, endometrium, kidneys, pancreas, prostate, liver, lungs, skin, thyroid gland, uterus, lymph nodes, tonsils, thymus, spleen, and bone marrow, and the second region includes areas of metastatic tumors in organs selected from the group consisting of: bone, lymph nodes, lungs, liver, brain, adrenal glands, breast, eyes, kidneys, muscles, pancreas, salivary glands, and spleen. In some embodiments, the first region includes a region of metastatic tumors in organs selected from the group consisting of: bone, lymph nodes, lung, liver, brain, adrenal glands, breast, eye, kidney, muscle, pancreas, salivary glands, and spleen, and the second region includes a region of primary tumors in organs selected from the group consisting of: breast, bladder, brain, colon, rectum, endometrium, kidney, pancreas, prostate, liver, lung, skin, thyroid gland, uterus, lymph nodes, tonsils, thymus, spleen, and bone marrow. In some embodiments, the first region includes identified tumors, and the second region does not include identified tumors.

[0014] In some embodiments, this document discloses a radiotherapy system comprising a radiation source providing an ablation dose and a subablation dose; and a processor coupled to the radiation source, wherein the processor is configured with the aforementioned instructions. In some embodiments, the ablation dose in a first region comprises between 20 and 100 Gy. In some embodiments, the ablation dose in the first region comprises between 20 and 60 Gy. In some embodiments, the subablation dose comprises between 0.1 and 2 Gy, and optionally, the subablation dose comprises multiple subablation doses, each of which in a second region comprises between 0.1 and 2 Gy. In some embodiments, the subablation dose comprises between 0.1 and 0.5 Gy, and optionally, the subablation dose comprises multiple subablation doses, each of which in a second region comprises between 0.1 and 5 Gy. In some embodiments, three subablation doses are administered. In some embodiments, more than three subablation doses are administered. In some embodiments, the first subablation dose is administered within 24 hours after the ablation dose is administered. In some embodiments, the first subablation dose is administered between 6 and 26 hours after the ablation dose. In some embodiments, the treatment reduces the size or intensity of the treated tumor, as measured by imaging selected from the group consisting of: computed tomography, magnetic resonance imaging, positron emission tomography, and computed tomography. In some embodiments, the treatment improves subject survival, reduces the number or severity of symptoms experienced by the subject, increases the number of immune cells in the tumor microenvironment, or increases the number of activated immune cells in the tumor microenvironment. In some embodiments, the radiation is selected from the group consisting of: X-ray radiation, gamma-ray radiation, alpha particle radiation, beta particle radiation, neutron particle radiation, external beam radiation, and brachytherapy.

[0015] Incorporation

[0016] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually incorporated by reference. Attached Figure Description

[0017] The novel features of the invention are set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description and accompanying drawings, which illustrate exemplary embodiments in which the principles of the invention are utilized, in which:

[0018] Figure 1 A radiotherapy system suitable for use with methods and procedures according to some embodiments of this disclosure is shown.

[0019] Figure 2A The present disclosure illustrates an object having a tumor in the liver, as an example of a tumor in the first region, according to some embodiments thereof.

[0020] Figure 2B The illustration shows an object to be treated in two regions according to some embodiments of the present disclosure, the two regions including a first region containing a first tumor and a second region containing a second tumor.

[0021] Figure 3 Examples of first, second, and third treatment regions in an object according to some embodiments of the present disclosure are shown, wherein the first treatment region includes a tumor in the lung, the second treatment region includes the entire lung, and the third treatment region includes a metastatic tumor.

[0022] Figure 4 A process for treating a patient according to some embodiments of this disclosure is shown.

[0023] Figure 5A and Figure 5B Two models for radioactive initiation according to some embodiments of this disclosure are shown. Figure 5A The study illustrates a pre-triggered treatment method in which four sub-ablation doses are delivered to the tumor to trigger the immune system before the ablation dose is delivered. Figure 5B This study illustrates a post-ablation modulation therapy method in which an ablation dose of radiotherapy is delivered to the tumor, followed by four subablation doses. The initial ablation dose activates effector T cells, while subsequent subablation doses alter the tumor microenvironment to increase immune infiltration, modify the cytokine environment, and reprogram macrophages.

[0024] Figure 6 A computer system used according to some embodiments of this disclosure is shown.

[0025] Figure 7A Treatment regimens for tumor growth are shown, comparing pre- and post-priming in mice carrying 3LL tumors with controls. * indicates p < 0.05, measured by log-rank (Mantel-Cox). Figures 7A-7D n = 5.

[0026] Figure 7B The relative tumor growth in the treatment group is shown.

[0027] Figure 7C The time it took to reach 3 times the initial tumor volume is shown.

[0028] Figure 7D The survival rate of the treated mice is shown.

[0029] Figure 8AThe schematic treatment regimens for local PAM and the treatment group are shown. n = 4-5 mice.

[0030] Figure 8B As shown Figure 8A Relative tumor growth curves in mice treated with the drug. n = 4-5 mice.

[0031] Figure 8C The time it took for treated mice carrying 3LL tumors to reach three times their initial volume is shown. n = 28–35 mice.

[0032] Figure 8D The composite survival curves in mice carrying 3LL tumors across multiple experiments are shown. * indicates p < 0.05, according to the Mantel-cox and Grehan-Breslow-Wilcoxon tests.

[0033] Figure 8E Tumor growth in nude mice carrying 3LL tumors is shown. n = 10-13 mice.

[0034] Figure 8F This represents the survival rate of nude mice carrying 3LL tumors. n = 10-13 mice.

[0035] Figures 9A-9C In vitro therapy involving 3LL tumor cells. Figure 9A A schematic diagram of an in vitro treatment regimen for 3LL tumor cells using PAM is shown. Figure 9B It shows that in such Figure 9A Cell death at 6 and 24 hours post-treatment was measured using LIVE / Dead fixed dye. Figure 9C It shows that in such Figure 9A Immunomodulatory, stress, and immunosuppressive markers were analyzed phenotypically by surface expression at 6 and 12 hours post-treatment. *p<0.05, **p<0.005, ***p<0.0005, and ****p<0.0001, determined by t-test.

[0036] Figure 9D -G involves in vitro treatment of a subset of the immune population with 0.5 Gyx4. Figure 9D The activity of a subset of T cells sorted after treatment was shown. Figure 9E The expression of CD25 and FOXP3 after treatment of sorted CD4+CD25+FOXP3+(Treg) is shown. Figure 9F The expression of CD206 (a marker of M2 macrophages) was shown in bone marrow-derived macrophages with cytokine polarization. Figure 9GThe results show cytokine secretion by bone marrow-derived macrophages with cytokine polarization after treatment. *p<0.05, **p<0.005, ***p<0.0005, and ****p<0.0001, determined by t-test.

[0037] Figure 10A A schematic diagram of the treatment and harvesting process for local PAM therapy in mice carrying 3LL tumors is shown.

[0038] Figure 10B The infiltrating leukocytes and Tregs in vivo, as measured by flow cytometry, are shown on days 6 and 10 after initiation of local PAM treatment. *p<0.05, **p<0.005, multiple comparisons by ANOVA.

[0039] Figure 10C RNA expression in whole tumor lysate of FOXP3 is shown on day 6. *p<0.05, **p<0.005, by ANOVA multiple comparisons.

[0040] Figure 10D The tumor infiltration of effector cells secreting granzyme B, as measured by flow cytometry, is shown on days 6 and 10.

[0041] Figure 10E The phenotypic analysis of tumor-infiltrating macrophage polarization by flow cytometry is shown on days 6 and 10 after treatment initiation. *p<0.05, **p<0.005, by ANOVA multiple comparisons.

[0042] Figure 11A The percentage of lymphocytes in the spleen, as determined by flow cytometry, is shown on days 6 and 10. *p<0.05, **p<0.005, all groups were compared by ANOVA multiple comparisons.

[0043] Figure 11B The percentage of lymphocytes in draining lymph nodes as determined by flow cytometry on days 6 and 10 is shown. *p<0.05, **p<0.005, all groups were compared by ANOVA multiple comparisons.

[0044] Figure 12A Characterization of CD8 T cells and Treg populations in draining lymph nodes on days 6 and 10 following local PAM treatment is shown. *p<0.05, **p<0.005, t-test, n=4–6.

[0045] Figure 12B Characterization of CD8 T cells and Treg populations in the spleen on days 6 and 10 after local PAM treatment is shown. *p<0.05, **p<0.005, t-test, n=4-6.

[0046] Figure 12C ELISPOT was shown on the secretion of functional cytokines of granzyme B from the spleen of treated mice on days 6 and 10. *p<0.05, t-test, n=4–6.

[0047] Figure 12D ELISPOT data on the functional cytokine secretion of IFNγ from the spleen of treated mice at days 6 and 10 are shown. *p<0.05, t-test, n=4–6.

[0048] Figure 13A Tumor measurements were shown in mice carrying orthotopic 4T1 tumors treated with local PAM and control mice. n = 12-13 mice.

[0049] Figure 13B The relative tumor growth in mice carrying 4T1 tumors treated with local PAM is shown. n = 12-13 mice.

[0050] Figure 13C Survival curves of 4T1 mice treated with local PAM are shown. n = 12-13 mice.

[0051] Figure 14A This diagram illustrates a systemic PAM treatment using whole-lung irradiation following ablation of the primary tumor.

[0052] Figure 14B The 2-month survival and overall survival rates of systemic PAM treatment compared to primary tumor ablation alone are shown. * indicates p<0.05, according to Mantel-cox and Grehan-Breslow-Wilcoxon tests. n = 26–27 mice.

[0053] Figure 14C The image shows lungs 12 days after Indian ink injection with and without whole-lung irradiation (red arrows indicate large metastases) and a diagram listing visible large metastases.

[0054] Figure 14D Histological sections of the lungs are shown (red asterisks indicate metastatic lesions) and diagrams of the listed lesions.

[0055] Figure 15A This shows a PET scan 28 days after ablation of the primary tumor. Seven images are represented. Figure 15B The images show PET scans 28 days after ablation of the primary tumor and 12 days after whole-lung irradiation. Six images are represented.

[0056] Figure 16AThe Treg population in the whole lung is shown after whole-lung PAM treatment. *p<0.05, **p<0.005, ANOVA multiple comparisons.

[0057] Figure 16B The whole lung phenotype analysis by flow cytometry is shown 19 days after ablation of the primary tumor. *p<0.05, multiple comparisons by ANOVA.

[0058] Figure 16C Characterization of GzB-secreting T cells is shown. *p<0.05, **p<0.005, ***p<0.0005, ANOVA multiple comparisons.

[0059] Figure 16D Histological staining of CD8 (brown) and FOXP3 (green) in metastatic lung lesions is shown (22x magnification).

[0060] Figure 16E Splenic CD45+ cells were measured by flow cytometry 19 days after ablation of the primary tumor. *p<0.05, ****p<0.0001, all groups were compared by ANOVA multiple comparisons.

[0061] Figure 16F The splenic CD3+ T cells were measured by flow cytometry 19 days after ablation of the primary tumor. *p<0.05, ****p<0.0001, all groups were compared by ANOVA multiple comparisons.

[0062] Figure 16G The expression of class II MHC in splenic monocytes and monocytes after treatment is shown. *p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001, ANOVA multiple comparisons.

[0063] Figure 17 The effects of PAM-RT on local and systemic immune regulation were demonstrated.

[0064] Figure 18A The survival rates of mice treated with no treatment, with a single ablation dose or radiotherapy, pre-ablation induction, or post-ablation modulation are shown according to some embodiments of this disclosure.

[0065] Figure 18B Tumor vascular system density in mice treated with no treatment, with a single ablation dose or radiotherapy, with post-ablation conditioning, or with only four subablation doses, according to some embodiments of this disclosure, is shown.

[0066] Figure 19ATumor growth in C57Bl6 mice that are untreated, treated with a single ablation dose of radiotherapy, or treated with post-ablation modulation are shown according to some embodiments of the present disclosure.

[0067] Figure 19B Tumor growth in nude mice, in which no treatment was administered, in radiotherapy with a single ablation dose, or in treatment with post-ablation modulation, is shown according to some embodiments of the present disclosure.

[0068] Figure 20A The survival rates of C57Bl6 mice under untreated, radiotherapy with a single ablation dose, or post-ablation modulated treatment are shown according to some embodiments of the present disclosure.

[0069] Figure 20B The survival rates of nude mice treated with no treatment, with a single ablation dose of radiotherapy, or with post-ablation conditioning are shown according to some embodiments of this disclosure.

[0070] Figure 21A Leukocyte infiltration in 3LL tumors of C57Bl6 mice that were untreated, treated with a single ablation dose of radiotherapy (24 Gy), treated with post-ablation modulation, or pre-priming therapy, is shown according to some embodiments of this disclosure. For mice treated with a single ablation therapy, leukocyte infiltration is shown one and five days post-treatment.

[0071] Figure 21B Leukocyte infiltration in 3LL tumors of C57Bl6 mice that were untreated, treated with a single ablation dose of radiotherapy (24 Gy), treated with post-ablation modulation, or pre-priming therapy, is shown according to some embodiments of this disclosure. For mice treated with a single ablation therapy, leukocyte infiltration is shown one and five days post-treatment.

[0072] Figure 21C Leukocyte infiltration in 4T1 tumors of C57Bl6 mice treated with a single ablation dose of radiotherapy (24 Gy), post-ablation modulation, or pre-priming therapy, according to some embodiments of this disclosure, is shown. For mice treated with a single ablation therapy, leukocyte infiltration is shown one and five days post-treatment.

[0073] Figure 22A The survival rates of mice treated with trabectedin alone, with a single ablation dose (24 Gy), with post-ablation adjustment (22 Gy + 4 x 0.5 Gy), with a single ablation dose and trabectedin, or with post-ablation adjustment and trabectedin, according to some embodiments of the present disclosure, are shown.

[0074] Figure 22B Tumor growth in mice treated with trabectedin according to some embodiments of the present disclosure is shown.

[0075] Figure 22C Tumor growth in mice treated with a single ablation dose of radiotherapy and trabectedine according to some embodiments of this disclosure is shown.

[0076] Figure 22D Tumor growth in mice treated with ablation modulation and trabectedine according to some embodiments of this disclosure is shown.

[0077] Figure 23 Experimental procedures according to some embodiments of this disclosure are illustrated. Briefly, mice are injected with tumor cells (e.g., 4T1 cells) into the fourth mammary fat pad. Approximately 8 days later, the tumor is treated with three ablation doses of radiotherapy (3 x 20 Gy) over three days and an anti-PD1 therapeutic agent or mediator. Twelve days later, some mice are treated with four doses of subablation radiotherapy (4 x 0.5 Gy) over four days and an anti-PD1 therapeutic agent or mediator.

[0078] Figure 24A The survival rates of mice treated with D90 cells and three ablation doses of radiotherapy; three ablation doses of radiotherapy and an anti-PD1 therapeutic agent; three ablation doses of radiotherapy and four subablation doses; or three ablation doses of radiotherapy, four subablation doses, and an anti-PD1 therapeutic agent, according to some embodiments of this disclosure, are shown.

[0079] Figure 24B The survival rates of mice treated with intravenous 4T1 and an anti-PD1 therapeutic agent, four subablation doses, or four subablation doses and an anti-PD1 therapeutic agent are shown according to some embodiments of the present disclosure.

[0080] Figure 25 The survival rates of mice treated with 4T1 cells and three ablation doses of radiotherapy, three ablation doses of radiotherapy and an anti-PD1 therapeutic agent, three ablation doses of radiotherapy and four subablation doses, or three ablation doses of radiotherapy, four subablation doses and an anti-PD1 therapeutic agent, according to some embodiments of this disclosure, are shown.

[0081] Figure 26 The results showed that Tregs, which led to a significant increase in the CD8 / Treg ratio in the whole lung, were significantly reduced after whole-lung PAM treatment. *p<0.05, ANOVA multiple comparisons. Detailed Implementation

[0082] The methods and apparatus of this disclosure can treat cancer with an ablation RT dose and a subsequent subablation RT dose. The ablation RT dose can generate an adaptive immune response, and the subsequent subablation RT treatment can affect the tumor microenvironment and may trigger the production of antigenic substances in cancer cells, which can lead to an immunogenic response against the cancer cells. This subablation dose can be used to generate an immune response in vulnerable tissues in a way that protects these vulnerable tissues, thereby keeping these tissues viable after treatment. The methods and apparatus of this disclosure are well-suited for combination with existing methods, compounds, and apparatus for treating cancer. For example, the ablation and subablation doses as described herein can be delivered using a radiotherapy system known to those skilled in the art. The radiotherapy system can be programmed using software instructions to treat tumors with ablation and subablation doses. The software instructions may include treatment planning software to control the location and timing of the ablation and subablation doses. Alternatively or in combination, radiotherapy may include brachytherapy. For example, a radioactive seed may be placed near a tumor such as a prostate tumor, and subablation treatment may be applied at a location away from the prostate using a radiotherapy machine. The methods and apparatus disclosed herein can be combined with existing methods, apparatus, and compounds (such as immunomodulatory therapies) for treating cancer.

[0083] Unbound by any particular theory, it is believed that ablation doses can release large amounts of immunogenic antigens from cancer cells, and subsequent treatment with subablation doses can lead to the presentation of similar antigens by cancer cells exposed to the subablation dose, thereby triggering an immune response. Furthermore, subsequent subablation dose treatment may act on the tumor microenvironment and reduce the extent to which the tumor provides immune-exempt sites.

[0084] Different radiation schedules and doses for tumors can have different immunomodulatory effects. Treatment plans longer than 7 days can be immunosuppressive, while a single ablation dose releasing a large amount of antigen can be immunogenic. This disclosure provides radiation protocols for treating solid tumors that result in enhanced immunogenicity and increased tumor accessibility to immune cells and therapeutic agents. In some embodiments, the radiation protocols described herein combine non-ablation immunoinitiation with subsequent ablation, termed "immunoinitiated ablation" (IPA), and can produce more effective in situ vaccines.

[0085] Existing therapies are often ineffective in the treatment of certain solid tumors, partly because the tumor microenvironment exhibits immune-immune sites and leads to resistance to chemotherapy and radiotherapy. Multiple factors contribute to an immunosuppressive tumor microenvironment, including tumor-favorable immune cells, pro-connective tissue proliferation responses, and disordered tumor angiogenesis. Tumors may contain a large number of immunosuppressive stromal cells, such as myeloid-derived suppressor cells (MDSCs), cancer-associated fibroblasts (CAFs), and tumor-associated macrophages (TAMs). TAMs and CAFs can be key players in producing excessive extracellular matrix (ECM) by synergistically inducing fibrotic responses (similar to the "wound healing" response after injury). Tumor-associated macrophages constitute a large portion of resident immune cells in pancreatic cancer, thereby influencing the suppression of infiltrative cytotoxic T cells, which are typically characterized as M1 (anti-tumorigenic) and M2 (pro-tumorigenic). Dendritic cells (DCs) are a very small population of immune cells, but those present in tumors often exhibit tolerogenicity and lead to immunosuppressive regulatory T cells (T cells). reg The induction of MHC and the suppression of cytotoxic T cells. Many tumor cells downregulate the expression of major histocompatibility complex (MHC) class I and components of antigen presentation mechanisms, which can fundamentally prevent MHC-peptide presentation, thereby evading recognition by immune effector cells. Promoting connective tissue proliferation produces fibrotic reticular structures, further hindering the accessibility of cytotoxic immune cells and separating tumor cells from blood vessels while reducing permeability.

[0086] Disorganized and inefficient tumor vascular systems can also function in the immunosuppressive tumor microenvironment, partly due to unregulated tumor growth. As the tumor progresses, angiogenesis often fails to keep pace with growth, thus hindering the efficient formation of structured vascular networks. Tumor vascular systems are also characterized by immature and leaky vessels, leading to increased interstitial pressures seen in many solid tumors, which can result in reduced extravasation of immune cells. This disorganization may contribute to chemotherapy and radiotherapy resistance due to increased hypoxia and reduced accessibility to drugs and immune infiltration.

[0087] Radiation therapy has a recognized role in local tumor control through direct cell death. Anecdotal evidence suggests that tumor ablation can produce responses at distant sites in rare cases, believed to be due to the induction of systemic immunity. Radiation has been shown to induce immunogenic cell death (ICD) and may be the first step in anti-tumor immunity.

[0088] Radiation-induced damage and cellular stress can promote the activation and maturation of dendritic cells (DCs). DCs are antigen-presenting cells (APCs) that engulf antigens from their environment and present them to T cells on class I or II MHC receptors. T cell activation depends not only on antigen presentation from DCs but also on co-stimulatory molecules and is one of the most important stages of adaptive-dependent antitumor immunity. T cell activation can often be ineffective due to the immunosuppressive tumor microenvironment. According to the implementation scheme described herein, utilizing the activation potential of radiation while limiting its negative effects can yield much more effective clinical outcomes.

[0089] Clinically, three dosing regimens are available for cancer treatment: conventional fractionation, subablative hypofractionation, and ablation hypofractionation (Table 1). These existing dosing regimens can be combined with subablation therapy according to certain implementation plans. Conventional fractionation in cancer treatment involves the delivery of many low-dose fractions over a longer period (more than 7 days) and is generally considered to have immunosuppressive effects, repeatedly killing any radiosensitive infiltrating immune cells. Subablative hypofractionation involves the delivery of larger, non-lethal doses over less than 7 days and has some immunomodulatory effects. While this regimen enhances the antitumor immune response, its cytotoxicity as a single treatment is less effective in controlling tumor growth. Ablation hypofractionation leads to direct cell death, releases large amounts of antigens, and can control local tumor growth. A disadvantage of single ablation fraction RT is the induction of a pro-tumor fibrotic response, which is partly due to the attraction of tumor-associated macrophages and immunosuppressive cells in the tumorigenic phenotype that secrete TGFβ to the tumor microenvironment, which can reduce the antitumor response. The effects of different forms of fractionated radiotherapy are summarized in Tables 1 and 2 and are suitable for combination according to certain implementation plans.

[0090] Treating tumors with a combination of ablation therapy (e.g., a single ablation therapy) and one or more subablation therapies can combine the benefits of ablation therapy, which generates antigens and kills cells, with the benefits of subablation therapy, which improves immune access to the tumor site and activates the immune system. Initial ablation therapy leads to increased tumor antigen release and upregulation of damage-associated molecular patterns and class I MHC on surviving tumor cells, thereby reversing tumor immune escape. Further immunomodulatory benefits can be provided by administering this treatment with one or more subablation doses over a short period (e.g., less than 7 days), including normalization of the vascular system; upregulation of damage-associated molecular patterns, class I MHC, and adhesion markers; and increased release of chemokines, attracting effector T cells to the tumor and reducing the influx of immunosuppressive regulatory T cells. The effects of these two therapies can be synergistic, producing a much greater therapeutic effect than would be expected with either therapy alone. Table 2 summarizes the hypothetical effects of this treatment.

[0091] Table 1. Radioframing schemes used clinically

[0092]

[0093] Table 2. Experimental Radiation Protocol

[0094]

[0095] Figure 1 A radiotherapy treatment system 10 is illustrated, which can deliver radiotherapy to a patient 14 as described herein. The radiotherapy system 10 may include one or more components of a number of existing systems suitable for incorporation according to embodiments disclosed herein. Examples of existing systems suitable for incorporation according to this disclosure include systems from Accuray, such as the CyberKnife, Radixact, and TomoTherapy treatment systems, and systems from Varian, such as the Edge radiosurgery system, TrueBeam radiotherapy system, Calypso extracranial tracking system, and intracranial tracking. The treatment system may include tracking and imaging systems to align with a patient having a tumor. Radiotherapy treatment may include photon-based radiotherapy, brachytherapy, electron beam therapy, proton, neutron, or particle therapy, or other types of treatment therapies. The treatment system 10 includes a digital processing unit 601 to control the beam energy level and dose delivered to the subject, for example, via stereotactic radiotherapy (STRT). The treatment system 10 may also include imaging components and systems known to those skilled in the art. The radiotherapy treatment system 10 includes a gantry 18 coupled to a computer to control beam placement, and other beam guiding devices may also be used. The gantry 18 can support a radiation module 22, which may include a radiation source 24 and a linear accelerator 26 coupled to a computer and operable to generate a radiation beam 30. Although the gantry 18 shown is a ring-shaped gantry, i.e., it extends through the entire 360° arc to form a complete ring or circle, other types of mounting arrangements can be used. For example, a C-type, partially ring-shaped gantry, or robotic arm can be used. Any other frame capable of positioning the radiation module 22 at various rotational and / or axial positions relative to the patient 14 can also be used. Furthermore, the radiation source 24 can travel in a path that does not conform to the shape of the gantry 18. For example, the radiation source 24 can travel in a non-circular path, even though the gantry 18 shown is generally circular.

[0096] Radiation module 22 may also include adjustment device 34 operable to modify or adjust radiation beam 30. Adjustment device 34 provides adjustment of radiation beam 30 and directs radiation beam 30 toward patient 14. Specifically, radiation beam 34 is directed toward a portion of the patient. Broadly speaking, said portion may include the entire body, or may be smaller than the entire body and may be defined by a two-dimensional area and / or a three-dimensional volume. The portion to which radiation is desired is an instance of region of interest. Region of interest 38 may include a first treatment site, a second treatment site, or a subsequent treatment site. Region of interest 38 may also include an edge surrounding or partially surrounding a target. Another type of region of interest is an area at risk of radiation injury. If a portion includes an area at risk of radiation injury, the radiation beam is preferably redirected from that area. Patient 14 may have more than one area receiving radiation therapy as described herein.

[0097] Figure 2A An example of a patient with a tumor in a first region (in this case, the liver) is illustrated. Using the methods described herein, this tumor can be treated with an ablation dose of radiotherapy applied to the entire tumor or a portion thereof, followed by one or more subablation doses of radiotherapy to the same tissue. In some cases, the methods of this disclosure may involve applying a first ablation dose of radiotherapy to the entire tumor or a portion thereof, followed by one or more subablation doses applied to another or more locations of the tumor or the subject. The subablation dose may be applied to the entire tumor or a portion thereof. If the ablation dose is applied to a portion of the tumor, the subablation dose may be applied to the same portion of the tumor or to different portions thereof.

[0098] Subablative doses of radiotherapy delivered after the ablation dose can enhance the immune response to the tumor and improve treatment response. For example, as... Figure 7A As shown, mice with tumors were treated with untreated, radiotherapy with a single ablation dose applied to the tumor, or radiotherapy with a single ablation dose applied to the tumor followed by four subablation doses. Mice receiving the ablation dose followed by the subablation doses had improved survival rates. Mice receiving the ablation and subablation doses in the reverse order (i.e., four subablation doses followed by a single ablation dose) had slightly higher survival rates than mice treated with a single ablation dose alone, but not as high as when the ablation dose was administered before the subablation doses. A similar approach could be used to treat patients (such as human and animal patients), for example, through appropriate software instructions on a radiotherapy system as described herein.

[0099] Figure 2BAn example of a patient is illustrated who has two tumors in an organ, such as the lung. These two tumors may include a first tumor and a second tumor. The first tumor may include a primary tumor, while the second tumor may include a metastatic or recurrent tumor. Alternatively, the first tumor may include a metastatic or recurrent tumor, while the second tumor may include a primary tumor. Using the methods described herein, the first tumor can be treated with an ablation dose of radiotherapy applied to the entire tumor or a portion thereof. Subsequently, the first and second tumors can be treated separately with one or more subablation doses of radiotherapy. Subsequent subablation doses of radiotherapy can enhance the immune response to the tumor and improve the treatment response as described herein. In this case, the second tumor may stop growing or regress even though no ablation dose has been applied. Using the methods described herein, a patient may have a first tumor and any number of secondary tumors. A first ablation dose of radiotherapy can be applied to the first tumor, followed by one or more subablation doses of radiotherapy applied to some or all of the secondary tumors. Alternatively, subablation doses of radiotherapy can be applied to the first tumor after the application of an ablation dose of radiotherapy. Secondary tumors may be in the same organ as the tumor treated with the ablation dose, or they may be in one or more different organs. In some cases, the primary tumor is selected based on its size, location, disease stage, or other clinically relevant characteristics.

[0100] Figure 3 An example patient with a tumor in the lung and metastatic tumors in the bone is shown. In some cases, the lung tumor can be treated with a single ablation dose of radiation. After the first dose, the entire lung can be treated with one or more subablation doses of radiation. While treating the entire lung with one or more subablation doses, the bone with metastatic tumors can also be treated with one or more subablation doses. In another example, the site of bone metastasis can be treated first with a single ablation dose of radiation. After this first dose, the bone metastasis and the lung tumor or the entire lung can be treated with one or more subablation doses.

[0101] In some cases, after treating the primary tumor with an ablation dose, one or more subablation doses may be applied to the entire body. For example, in some cases, one or more subablation doses may be applied to the entire peritoneal cavity. In some cases, one or more subablation doses may be applied to an entire organ known to contain one or more metastatic tumors, or to an entire organ in a site known to potentially develop metastatic tumors. For example, a patient with breast cancer may develop metastatic tumors in the bone, lung, liver, or brain. A patient with lung cancer may develop metastatic tumors in the other lung or adrenal gland, bone, brain, and liver. Examples of organs that may develop metastatic tumors include, but are not limited to, bone, lymph nodes, lungs, liver, brain, adrenal glands, breast, eye, kidney, muscle, pancreas, salivary glands, and spleen.

[0102] Figure 4 An exemplary process 400 is illustrated, which can be used to treat cancer. The first step 405 of the process includes identifying a first treatment region. The first treatment region may include a tumor region within the patient. In some cases, the first treatment region may include a primary tumor. In some cases, the first treatment region may be the location of a metastatic tumor. In some cases, if several different tumors are present in the patient, the first treatment region may include the tumor location most easily radiotreated. In some cases, if several different tumors are present in the patient, the first treatment region may be the tumor location safest for radiotherapy. The tumor safest for radiotherapy may be a tumor located in a way that reduces the adverse effects of radiation compared to other locations. In some cases, the first treatment region includes the location of the largest tumor in the patient. In some cases, the first treatment region includes the location of the only known tumor in the patient.

[0103] After the first treatment region has been identified, a second treatment region is identified in step 410. In some embodiments, the second treatment region may include a location with a tumor. In some cases, the second treatment region may be a different region from the first treatment region. In some cases, the second treatment region may include the same region as the first treatment region. In some cases, the second treatment region may be in the same organ as the first treatment region or in a different organ. The second treatment region may be a location with a secondary or primary tumor. In some cases, the second treatment region has a tumor smaller than that in the first treatment region. In some cases, the second treatment region is a location that is difficult to treat with radiotherapy. In some cases, the second treatment region is a location that is more sensitive to the adverse effects of radiotherapy compared to the first treatment region. In some cases, the second treatment region is a location without the identified tumor, but it is known to be a region that may develop into a metastatic tumor.

[0104] The next step 415 includes registering the radiation therapy system with the patient to target the first treatment region. The radiation therapy system may include components of any radiation therapy system known in the art, such as known radiation therapy systems commercially available from Varian Medical and Accuray. For example, the radiation therapy system may include… Figure 1 Systems, or similar systems. Types of radiation therapy can include any type suitable for clinical use as described herein, such as X-ray radiation, gamma-ray radiation, alpha particle radiation, beta particle radiation, neutron particle radiation, external beam radiation, or brachytherapy. For example, a radiation therapy system can include an imaging system capable of imaging and targeting tumors within a patient.

[0105] Once the first treatment area is registered, it can be treated with an ablation dose of radiation in step 420. The ablation dose can be between about 10 Gy and about 60 Gy, about 20 Gy and about 40 Gy, or about 20 Gy and about 30 Gy. In some cases, the ablation dose is about 10 Gy, 12 Gy, 14 Gy, 16 Gy, 18 Gy, 20 Gy, 22 Gy, 24 Gy, 26 Gy, 28 Gy, 30 Gy, 32 Gy, 34 Gy, 36 Gy, 38 Gy, 40 Gy, 42 Gy, 44 Gy, 46 Gy, 48 Gy, 50 Gy, 52 Gy, 54 Gy, 56 Gy, 58 Gy, or 60 Gy, or within the range defined by any two of the above values.

[0106] Following the first treatment, step 425 of process 400 involves waiting for a predetermined period of time before proceeding with subsequent treatments. For example, the predetermined period of time may be between approximately 1 hour and approximately 4 days. In some cases, the predetermined period of time may be between approximately 1 hour and approximately 36 hours, approximately 6 hours and approximately 30 hours, approximately 12 hours and approximately 26 hours, approximately 20 hours and approximately 28 hours, or approximately 22 hours and approximately 26 hours. Those skilled in the art can determine an appropriate amount of waiting time based on the teachings of this disclosure.

[0107] In step 430, the radiotherapy system is registered at the second treatment region, and in step 435 the second treatment region is treated with a subablation dose. The subablation dose may include doses between: approximately 0.1 Gy and approximately 3 Gy, approximately 0.2 Gy and approximately 2 Gy, approximately 0.3 Gy and approximately 1 Gy, approximately 0.3 Gy and approximately 0.7 Gy, approximately 0.1 Gy and 0.5 Gy, or approximately 0.8 Gy and approximately 1.2 Gy. Steps 425 through 435 may be repeated multiple times. In some cases, steps 425-435 may be repeated two, three, four, five, six, or more than six times to induce an immunogenic response at the second treatment region or in another subablation treatment region without ablation. In some cases, the subablation dose includes multiple subablation doses, and each of the multiple subablation doses includes a dose between: about 0.1 Gy and about 3 Gy, about 0.2 Gy and about 2 Gy, about 0.3 Gy and about 1 Gy, about 0.3 Gy and about 0.7 Gy, about 0.1 Gy and 0.5 Gy, or about 0.8 Gy and about 1.2 Gy. Next, the radiotherapy system can be registered at the third treatment region in step 440, and the third treatment region can be treated with the subablation dose in step 445. The subablation dose can be a dose between: about 0.1 Gy and about 3 Gy, about 0.2 Gy and about 2 Gy, about 0.3 Gy and about 1 Gy, about 0.3 Gy and about 0.7 Gy, or about 0.8 Gy and about 1.2 Gy. If it is advantageous to induce an immunogenic response in the third treatment area without ablation, then steps 440 and 445 can be repeated any number of times.

[0108] After treatment in the third treatment area, the system waits for a predetermined time period 450, and then steps 440-450 can be repeated once, twice, three times, four times, or more than four times. The predetermined time period can be between approximately 1 hour and approximately 4 days. In some cases, the predetermined time period can be between: approximately 1 hour and approximately 36 hours, approximately 6 hours and approximately 30 hours, approximately 12 hours and approximately 26 hours, approximately 20 hours and approximately 28 hours, or approximately 22 hours and approximately 26 hours.

[0109] The processor described herein may be configured with instructions to execute method 400.

[0110] although Figure 4 A method 400 for treating cancer according to one embodiment is illustrated, but those skilled in the art will recognize many adaptations and variations. One or more steps may be omitted, repeated, performed simultaneously, and / or performed in a different order. In some embodiments, one or more steps may be modified, or may include sub-steps. Furthermore, those skilled in the art will understand that additional steps may be included when performing this method.

[0111] In some cases, the cumulative dose of radiotherapy delivered to the second region throughout the treatment process includes less than the ablation dose. In some cases, the cumulative subablation dose includes multiple subablation doses.

[0112] Figure 5A This illustration shows the biological processes that can be obtained by pre-inducing a tumor with one or more subablative doses of radiotherapy prior to the application of an ablation dose, according to some implementation schemes. In this case, a tumor displaying angiogenesis and immune cells (black circles) is first treated with four subablative doses (such as 0.5 Gy or 1 Gy, indicated by small “lightning bolts”), followed by a single ablation dose (such as 22 Gy or 20 Gy, indicated by large “lightning bolts”). The pre-inducing subablative doses sensitize the tumor to immune cells. This can be demonstrated by increased expression of stress markers, immunomodulatory / immunogenic cell death, and differences in peptide library and class I MHC expression. The tumor is then treated with the ablation dose of radiotherapy, which leads to cell death, particularly of tumor cells, and releases tumor antigens into the tumor microenvironment and circulation. Ablation radiotherapy also results in other changes in the surviving cells of the tumor, such as increased expression of damage-associated molecular patterns and increased release of cytokines, which are pro-immunogenic. The release of tumor antigens stimulates effector T cell responses, which are enhanced by tumor pre-sensitization achieved through subablation doses.

[0113] Figure 5B An example of post-ablation modulation and the biological processes involved is illustrated. In this case, a tumor displaying blood vessels and immune cells (black circles) is first treated with a single ablation dose (such as 22 Gy or 20 Gy, indicated by large “lightning bolts”), followed by four subablation doses (such as 0.5 Gy or 1 Gy, indicated by small “lightning bolts”). The initial ablation dose of radiation induces cell death and releases antigens that stimulate an effector T cell response. Subsequent subablation doses of radiation modulate the tumor environment and increase the infiltration of activated effector T cells into the tumor. Tumor changes induced by subablation treatment promote the increased infiltration; these changes include the normalization of the vascular system (see [link to relevant documentation]). Figure 7B ), altered cytokine environment and macrophage repolarization / reprogramming. For example... Figure 7A As shown, both pre-initiation treatment and post-ablation modulation improved the survival rate of tumor-bearing mice. However, post-ablation modulation showed a greater impact on survival. Figure 17 The effects of PAM-RT treatment on local and systemic immune regulation were summarized.

[0114] The methods and systems disclosed herein can be used to treat patients. In some cases, the patient is a person. In some cases, the patient has been diagnosed with cancer. In some cases, the patient has been diagnosed with solid cancer. Examples of cancer types include, but are not limited to: esophageal cancer, breast cancer, gastric cancer, bile duct cancer, pancreatic cancer, colon cancer, lung cancer, thymic cancer, mesothelioma, ovarian cancer, and endometrial cancer. In some cases, the first region may include the region of the organ's primary tumor. Examples of organs that can develop cancer include, but are not limited to, the breast, bladder, brain, colon, rectum, endometrium, kidney, pancreas, prostate, liver, lung, skin, thyroid, uterus, lymph nodes, tonsils, thymus, spleen, and bone marrow. In some cases, the patient may have more than one different type of cancer. In some cases, the patient has at least one detected tumor. In some cases, the patient's cancer is unresponsive to another therapy or has stopped responding to another therapy.

[0115] In some embodiments, this document describes a method for treating cancer in a subject, the method comprising delivering an ablation dose of radiation to a first region containing the cancer, followed by a subablation dose to a second region, wherein the subablation dose is administered after the ablation dose. In some embodiments, the subablation dose is administered at least 1 hour after the ablation dose. In some embodiments, the subablation dose is administered at least 1 day after the ablation dose. In some embodiments, the subablation dose is administered no more than 4 days after the ablation dose.

[0116] Digital processing device

[0117] In some embodiments, the platforms, systems, media, and methods described herein include digital processing devices or their use. In further embodiments, the digital processing device includes one or more hardware central processing units (CPUs), general-purpose graphics processing units (GPGPUs), or field-programmable gate arrays (FPGAs) that perform the functions of the device. In even further embodiments, the digital processing device also includes an operating system configured to execute executable instructions. In some embodiments, the digital processing device is optionally connected to a computer network. In even further embodiments, the digital processing device is optionally connected to the Internet, enabling it to access the World Wide Web. In even further embodiments, the digital processing device is optionally connected to a cloud computing facility. In other embodiments, the digital processing device is optionally connected to an intranet. In other embodiments, the digital processing device is optionally connected to a data storage device.

[0118] Based on the description herein, and by way of non-limiting example, suitable digital processing devices include server computers, desktop computers, laptop computers, notebook computers, mini-notebook computers, netbook computers, internet tablet computers, set-top box computers, streaming media devices, handheld computers, internet devices, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and carriers. Those skilled in the art will recognize that many smartphones are suitable for use in the systems described herein. Those skilled in the art will also recognize that selected televisions, video players, and digital music players with optional computer network connectivity are suitable for use in the systems described herein. Suitable tablet computers include those with netbooks, tablets, and convertible configurations known to those skilled in the art.

[0119] In some embodiments, the digital processing device includes an operating system configured to execute executable instructions. An operating system is, for example, software containing programs and data that manages the device's hardware and provides services for executing applications. Those skilled in the art will recognize that, by way of non-limiting examples, suitable server operating systems include FreeBSD, OpenBSD, etc. Linux Mac OS X Windows as well as Those skilled in the art will recognize that, by way of non-limiting example, suitable personal computer operating systems include Mac OS and such UNIX-like operating systems. In some implementations, the operating system is provided by cloud computing. Those skilled in the art will also recognize, by way of non-limiting example, suitable mobile smartphone operating systems include... OS Research In BlackBerry Windows OS Windows OS as well as Those skilled in the art will also recognize that, by way of non-limiting example, suitable streaming device operating systems include Apple. Google Google Amazon and Those skilled in the art will also recognize, by way of non-limiting example, that suitable video game console operating systems include: Xbox Xbox One and

[0120] In some embodiments, the apparatus includes a storage and / or memory device. The storage and / or memory device is one or more physical devices for temporarily or permanently storing data or programs. In some embodiments, the device is volatile memory and requires power to maintain the stored information. In some embodiments, the device is non-volatile memory and retains the stored information when the digital processing device is not powered. In further embodiments, the non-volatile memory includes flash memory. In some embodiments, the non-volatile memory includes dynamic random access memory (DRAM). In some embodiments, the non-volatile memory includes ferroelectric random access memory (FRAM). In some embodiments, the non-volatile memory includes phase-change random access memory (PRAM). In other embodiments, the apparatus is a storage device, which, by non-limiting example, includes CD-ROMs, DVDs, flash memory devices, disk drives, magnetic tape drives, optical disc drives, and cloud-based storage. In further embodiments, the storage and / or memory device is a combination of those devices disclosed herein.

[0121] In some embodiments, the digital processing device includes a display that transmits visual information to a user. In some embodiments, the display is a cathode ray tube (CRT). In some embodiments, the display is a liquid crystal display (LCD). In a further embodiment, the display is a thin-film transistor liquid crystal display (TFT-LCD). In some embodiments, the display is an organic light-emitting diode (OLED) display. In various further embodiments, the OLED display is a passive-matrix OLED (PMOLED) or an active-matrix OLED (AMOLED) display. In some embodiments, the display is a plasma display. In other embodiments, the display is a video projector. In even further embodiments, the display is a combination of devices such as those disclosed herein.

[0122] In some embodiments, the digital processing device includes an input device for receiving information from a user. In some embodiments, the input device is a keyboard. In some embodiments, the input device is a pointing device, including, by non-limiting examples, a mouse, trackball, trackpad, joystick, game controller, or stylus. In some embodiments, the input device is a touchscreen or multi-touch screen. In other embodiments, the input device is a microphone for capturing voice or other sound input. In other embodiments, the input device is a camera or other sensor for capturing motion or visual input. In further embodiments, the input device is Kinect, Leap Motion, or the like. In further embodiments, the input device is a combination of devices such as those disclosed herein.

[0123] See Figure 6 In certain embodiments, exemplary digital processing device 601 is programmed or otherwise configured as a radiotherapy device as described herein. Device 601 can regulate various aspects of the radiotherapy device of this disclosure, such as performing processing steps. In this embodiment, digital processing device 601 includes a central processing unit 605 (CPU, also referred to herein as a “processor” and “computer processor”), which may be a single-core or multi-core processor or multiple processors for parallel processing. Digital processing device 601 also includes memory or memory location 610 (e.g., random access memory, read-only memory, flash memory), electronic storage unit 615 (e.g., hard disk), communication interface 620 for communicating with one or more other systems (e.g., network adapter), and peripheral devices 625, such as caches, other memories, data storage, and / or electronic display adapters. Memory 610, storage unit 615, interface 620, and peripheral devices 625 communicate with CPU 605 via a communication bus (solid line), such as a motherboard. Storage unit 615 may be a data storage unit (or data repository) for storing data. Digital processing device 601 can be operatively coupled to computer network (“network”) 630 via communication interface 620. Network 630 can be the Internet, the Internet and / or an extranet, or an intranet and / or extranet communicating with the Internet. In some cases, network 630 is a telecommunications and / or data network. Network 630 may include one or more computer servers that can support distributed computing such as cloud computing. In some cases, network 630 can implement a peer-to-peer network via device 601, which allows devices coupled to device 601 to act as clients or servers.

[0124] Continue to refer to Figure 6The CPU 605 can execute a series of machine-readable instructions, which can be embodied in a program or software. The instructions can be stored in a storage location, such as memory 610. The instructions can be directed to the CPU 605, which can then be programmed or otherwise configured to implement the methods of this disclosure. Examples of operations performed by the CPU 605 can include reading, decoding, executing, and writing back. The CPU 605 can be part of circuitry such as an integrated circuit. The circuitry can include one or more other components of the device 601. In some cases, the circuitry is an application-specific integrated circuit (ASIC) or a field-programmable gate array (FPGA).

[0125] Continue to refer to Figure 6 Storage unit 615 can store files, such as drives, libraries, and saved programs. Storage unit 615 can store user data, such as user preferences and user programs. Digital processing device 601 may, in some cases, include one or more additional data storage units located externally, such as on a remote server communicating via an intranet or the Internet.

[0126] Continue to refer to Figure 6 The digital processing device 601 can communicate with one or more remote computer systems via network 630. For example, device 601 can communicate with a user's remote computer system. Examples of remote computer systems include personal computers (e.g., portable PCs), touchscreen or tablet PCs (e.g., tablet PCs), and more. iPad Galaxy Tab), telephone, smartphone (e.g., iPhone, Android-enabled devices (or personal digital assistant)

[0127] The methods described herein can be implemented by means of machine-executable code stored in an electronic storage location of digital processing device 601, such as memory 610 or electronic storage unit 615. The machine-executable or machine-readable code can be provided in software form. During use, the code can be executed by processor 605. In some cases, the code can be retrieved from storage unit 615 and stored in memory 610 for access by processor 605. In some cases, electronic storage unit 615 can be excluded, and the machine-executable instructions can be stored in memory 610.

[0128] In some embodiments, this document discloses a computer-readable medium configured with instructions that, when executed, cause a processor to instruct a radiotherapy system to deliver an ablation dose of radiotherapy to a first region, and then, subsequently, a subablation dose to a second region. In some embodiments, the subablation dose is delivered at least one hour after the ablation dose. In some embodiments, the subablation dose is delivered at least one day after the ablation dose. In some embodiments, the subablation dose is delivered no more than four days after the ablation dose.

[0129] Non-transitory computer-readable storage medium

[0130] In some embodiments, the platforms, systems, media, and methods disclosed herein include one or more non-transitory computer-readable storage media encoded with programs comprising instructions executable by an operating system of an optionally networked digital processing device. In further embodiments, the computer-readable storage medium is a tangible component of the digital processing device. In still further embodiments, the computer-readable storage medium may optionally be removable from the digital processing device. In some embodiments, by way of non-limiting example, the computer-readable storage medium includes CD-ROMs, DVDs, flash memory devices, solid-state storage, disk drives, magnetic tape drives, optical disc drives, cloud computing systems and services, etc. In some cases, the programs and instructions are permanently, substantially permanently, semi-permanently, or non-transitory encoded on the medium.

[0131] Computer program

[0132] In some implementations, the platforms, systems, media, and methods disclosed herein include at least one computer program or its use. A computer program contains a sequence of instructions executable in the CPU of a digital processing device and written to perform a specified task. Computer-readable instructions may be implemented as program modules that perform a specific task or implement a specific abstract data type, such as functions, objects, application programming interfaces (APIs), data structures, etc. In view of the disclosure provided herein, those skilled in the art will recognize that computer programs can be written in various versions of various languages.

[0133] In various environments, the functionality of computer-readable instructions can be combined or assigned as needed. In some embodiments, the computer program includes a sequence of instructions. In some embodiments, the computer program includes multiple sequences of instructions. In some embodiments, the computer program is provided from one location. In other embodiments, the computer program is provided from multiple locations. In various embodiments, the computer program includes one or more software modules. In various embodiments, the computer program includes, in part or in whole, one or more web applications, one or more mobile applications, one or more standalone applications, one or more web browser plugins, extensions, add-ons, or add-ons, or combinations thereof.

[0134] Network Applications

[0135] In some implementations, the computer program includes a web application. Given the disclosure provided herein, those skilled in the art will recognize that, in various implementations, the web application utilizes one or more software frameworks and one or more database systems. In some implementations, in such... Web applications are created on the .NET or Ruby on Rails (RoR) software framework. In some implementations, the web application utilizes one or more database systems; as non-limiting examples, database systems include relational database systems, non-relational database systems, object-oriented database systems, relational database systems, and XML database systems. In further implementations, as non-limiting examples, suitable relational database systems include... SQL Server, MySQL TM and Those skilled in the art will also recognize that, in various embodiments, the web application is written in one or more versions of one or more languages. Web applications can be written in one or more markup languages, presentation definition languages, client-side scripting languages, server-side coding languages, database query languages, or combinations thereof. In some embodiments, the web application is written to some extent in a markup language such as Hypertext Markup Language (HTML), Extensible Hypertext Markup Language (XHTML), or Extensible Markup Language (XML). In some embodiments, the web application is written to some extent in a presentation definition language such as Cascading Style Sheets (CSS). In some embodiments, the web application is written to some extent in a language such as Asynchronous JavaScript and XML (AJAX). Actionscript, Javascript or They are written in client-side scripting languages ​​such as Active Server Pages (ASP). In some implementations, web applications are, to some extent, based on client-side scripting languages ​​such as Active Server Pages (ASP). Perl, Java TM Java Server Pages (JSP), Hypertext Preprocessor (PHP), Python™, Ruby, Tcl, Smalltalk, It can be written in server-side coding languages ​​such as Groovy. In some implementations, the web application is written to some extent in a database query language such as Structured Query Language (SQL). In some implementations, the web application integrates technologies such as... Lotus Enterprise server products, etc. In some implementations, the network application includes a media player element. In various further implementations, the media player element utilizes one or more of a number of suitable multimedia technologies; for example, multimedia technologies include... HTML 5 Java TM as well as

[0136] Mobile application

[0137] In some embodiments, the computer program includes a mobile application provided to a mobile digital processing device. In some embodiments, the mobile application is provided to the mobile digital processing device upon completion of manufacturing. In other embodiments, the mobile application is provided to the mobile digital processing device via the computer network described herein.

[0138] Given the disclosure provided herein, mobile applications are created using techniques known to those skilled in the art, utilizing hardware, languages, and development environments known in the art. Those skilled in the art will recognize that mobile applications are written in several languages. For non-limiting examples, suitable programming languages ​​include C, C++, C#, Objective-C, and Java. TM ,Javascript,Pascal,Object Pascal,Python TM Ruby, VB.NET, WML, and XHTML / HTML or a combination thereof with or without CSS.

[0139] Suitable mobile application development environments are available from several sources. As non-limiting examples, commercially available development environments include AirplaySDK, alcheMo, etc. Celsius, Bedrock, Flash Lite, the .NET Compact Framework, Rhomobile, and the WorkLight mobile platform are all available. Other development environments are freely available, including, for example, Lazarus, MobiFlex, MoSync, and Phonegap. Additionally, mobile device manufacturers distribute software development kits, including, for example, the iPhone and iPad (iOS) SDKs and Android SDKs. TM SDK SDK, BREW SDK, OS SDK, Symbian SDK, webOS SDK and Mobile SDK.

[0140] Those skilled in the art will recognize that several business forums can be used to distribute mobile applications, including, to name a few, non-limiting examples. App Store Play, Chrome Web Store AppWorld, Palm devices' App Store, webOS's App Catalog Marketplace for Mobile Ovi Store device Apps and DSi Shop.

[0141] standalone application

[0142] In some implementations, the computer program includes a standalone application, which is a program that runs as an independent computer process, rather than an appendage to an existing process, e.g., not a plugin. Those skilled in the art will recognize that standalone applications are typically compiled. A compiler is a computer program that can translate source code written in a programming language into binary object code, such as assembly language or machine code. By way of non-limiting examples, suitable compiled programming languages ​​include C, C++, Objective-C, COBOL, Delphi, Eiffel, and Java. TM Lisp, Python TM Visual Basic and VB.NET, or a combination thereof. Compilation is typically performed at least partially to create an executable program. In some implementations, the computer program includes one or more executable applications.

[0143] Web browser plugin

[0144] In one embodiment, the computer program includes web browser plugins (e.g., extensions, etc.). In computing, a plugin is one or more software components that add specific functionality to a larger software application. Software application manufacturers support plugins to enable third-party developers to create applications that can be extended, easily add new features, and reduce the application size. When supported, plugins allow for customization of the software application's functionality. For example, plugins are commonly used in web browsers to play videos, generate interactivity, scan for viruses, and display specific file types. Those skilled in the art will be familiar with several web browser plugins, including... Player and In some implementations, the toolbar includes one or more web browser extensions, add-ons, or supplements. In some implementations, the toolbar includes one or more browser bars, toolbars, or desktop toolbars.

[0145] Given the disclosure provided herein, those skilled in the art will recognize that several plugin frameworks can be used to support plugin development in various programming languages, including, to name a few, C++, Delphi, and Java. TM PHP, Python TM And VB.NET, or a combination thereof.

[0146] A web browser (also known as an internet browser) is a software application designed for use with a digital processing device connected to a network to retrieve, display, and traverse information resources on the World Wide Web. As a non-limiting example, suitable web browsers include... Internet Chrome Opera And KDE Konqueror. In some implementations, the web browser is a mobile web browser. A mobile web browser (also known as a microbrowser, mini-browser, and wireless browser) is designed for use on mobile digital processing devices, including, by way of non-limiting examples, handheld computers, tablet computers, netbook computers, small notebook computers, smartphones, music players, personal digital assistants (PDAs), and handheld video game systems. By way of non-limiting examples, suitable mobile web browsers include... Browser, RIM Browser Blazer, Browser move, Internet move, Basic Web, Browser, Opera move and PSP TM Browser.

[0147] Software Module

[0148] In some embodiments, the platforms, systems, media, and methods disclosed herein include software, server, and / or database modules or their use. In view of the disclosure provided herein, software modules are created using machines, software, and languages ​​known in the art, and through techniques known to those skilled in the art. The software modules disclosed herein are implemented in a variety of ways. In various embodiments, a software module includes files, code segments, programming objects, program structures, or combinations thereof. In further embodiments, a software module includes multiple files, multiple code segments, multiple programming objects, multiple program structures, or combinations thereof. In various embodiments, by way of non-limiting example, one or more software modules include web applications, mobile applications, and standalone applications. In some embodiments, the software module is within a computer program or application. In other embodiments, the software module is within more than one computer program or application. In some embodiments, the software module is hosted on a single machine. In other embodiments, the software module is hosted on more than one machine. In further embodiments, the software module is hosted on a cloud computing platform. In some embodiments, the software module is hosted on one or more machines in one location. In other embodiments, the software module is hosted on one or more machines in more than one location.

[0149] database

[0150] In some embodiments, the platforms, systems, media, and methods disclosed herein include one or more databases or their use. In view of the disclosure provided herein, those skilled in the art will recognize that many databases are suitable for storing and retrieving information. In various embodiments, by way of non-limiting example, suitable databases include relational databases, non-relational databases, object-oriented databases, object databases, entity-relational model databases, association databases, and XML databases. Further non-limiting examples include SQL, PostgreSQL, MySQL, Oracle, DB2, and Sybase. In some embodiments, the database is Internet-based. In further embodiments, the database is network-based. In still further embodiments, the database is cloud-based. In other embodiments, the database is based on one or more local computer storage devices.

[0151] Example 1 Local PAM-RT slows tumor growth and improves survival rate in mice

[0152] To investigate the efficacy of PAM-RT in local tumor control, C57BL / 6 mice with palpable subcutaneous 3LL tumors were divided into five treatment cohorts for preliminary study—untreated, 24 Gy on day 1 or day 5 of treatment, 1 Gy x 4 followed by 20 Gy, or 20 Gy followed by 1 Gy x 4. Essentially, four 1 Gy doses delivered to the primary tumor before or after a single 20 Gy dose were compared to a single 24 Gy RT dose. Pretreatment with low-dose RT showed minimal tumor control compared to a single RT dose, but significant growth delay and survival improvement were observed after 1 Gy x 4 doses of PAM-RT. Figure 7A -D is shown.

[0153] Because 3LL tumors grow rapidly, the efficacy of RT induction on day 5 of treatment may be affected by differences in tumor size at the time of ablation RT. Further studies on tumor growth yielded a 0.5 Gy x 4 fractions of PAM-RT as the optimal modulating dose for all further experiments. As previously mentioned, a single 24 Gy ablation fraction was compared to 22 Gy followed by four 0.5 Gy fractions of PAM-RT. A schematic of the dosing schedule is shown below. Figure 8A In contrast, PAM-RT delayed tumor growth (e.g., compared to 24 Gy). Figure 8B (as seen in the growth curve) and the time to reach three times the volume ( Figure 8C (As shown in the image). Furthermore, PAM-RT significantly improved the survival rate of these animals, such as... Figure 8D As observed, although a single 24 Gy dose could not cure 3LL tumor growth, a significant growth delay was observed in mice carrying large 3LL tumors after PAM-RT, while the effect of a single ablation RT dose was minimal. Finally, the effect of PAM-RT disappeared in immunocompromised (naked) mice. Figure 8E Tumor growth in nude mice carrying 3LL tumors was demonstrated, and Figure 8F The survival rates of these mice are shown. These data indicate that PAM can delay local tumor progression and significantly improve the survival rate of mice carrying 3LL tumors, although all mice eventually died from local tumor growth. Furthermore, the effectiveness of PAM-RT depends on a capable immune system.

[0154] Immunomodulatory effects of local PAM-RT in vitro

[0155] The immunomodulatory effect of PAM-RT on 3LL cells was also studied in vitro. The experimental protocol is shown below. Figure 9A Although no difference in cell death was observed 6 hours post-treatment, PAM-RT induced significantly more cell death at 24 hours than ablation alone. Figure 9BCompared to untreated cells and cells treated with low-dose radiation, ablation radiation with or without PAM-RT increased cell surface immunomodulatory markers (CD80), stress markers (CRT, Hsp70, Fas, and MHCI), and immunosuppressive markers (CD47 and PD-L1) at 6 hours post-RT. Interestingly, at 24 hours, PAM-RT-treated cells showed higher cell surface expression of 4-1BBL, CRT, Fas, Hsp70, and PD-L1 compared to ablation alone. Figure 9C As shown.

[0156] To examine the effects of PAM-RT on immune cells in vitro, bone marrow-derived macrophages and spleen T cells were collected from wild-type C57Bl / 6 mice. Macrophages were either polarized to M1 or M2 by cytokines or untreated. Splenic T cells were sorted into three major T cell populations: CD8+, CD4+CD25-, and CD4+CD25+ (Treg). One day after polarization or sorting, all populations were treated with a low dose of RT (0.5 Gy x 4). Due to the radiosensitivity of immune cells, the ablation dose was not included in the treatment. Characterization of the T cell populations 6 hours after the last dose showed a significant decrease in Treg activity, which was not observed in the CD8 or CD4+CD25- T cell populations. See [link to relevant documentation]. Figure 9D In the sorted Treg population, cells treated with low-dose RT showed a trend toward reduced CD25 surface expression and a significant reduction in both FOXP3 and CD25 dual expression, such as... Figure 9E As shown. Figure 9F As shown, M2-polarized macrophages treated with low-dose RT exhibited a significant reduction in CD206 expression (i.e., the M2 marker). M2-polarized macrophages also tended to express less IL-10 after RT, indicating repolarization following treatment. See [link to relevant documentation]. Figure 9G These data demonstrate that PAM-RT enhances the cytotoxicity of ablation RT without suppressing immunomodulatory effects. Furthermore, our low-dose RT reduces Tregs and reprograms M2 macrophages to a less immunosuppressive phenotype.

[0157] Local PAM-RT promotes tumor microenvironment remodeling by reducing Treg and M2 macrophages.

[0158] To investigate the immunological effects of PAM-RT on the tumor microenvironment (TME), irradiated 3LL tumors were collected on days 6 and 10 after the start of RT. Figure 10A As shown. Compared with ablation RT alone, leukocyte infiltration in tumors treated with PAM-RT was significantly increased. After phenotypic analysis of infiltrating leukocytes, intratumoral Tregs were significantly reduced in mice treated with PAM-RT on day 6 compared with ablation RT alone (see...). Figure 10B And the CD8 / Treg ratio increased significantly. Figure 10C As observed, RT-PCR of whole tumor RNA showed a significant reduction in FOXP3 mRNA expression in tumors treated with PAM-RT on day 6. Figure 10D This remodeling was observed to be accompanied by a trend of increased intratumoral CD8+ T cells secreting granzyme B on days 6 and 10, indicating enhanced effector CTL responses. Characterization of the intratumoral bone marrow cell population revealed a significant decrease in IL-10-secreting macrophages and a slight decrease in CD206 expression on day 6, and a trend towards decreased IL-10 secretion and significantly decreased CD206 expression on day 10 after treatment initiation. Figure 10E As shown. In summary, PAM-RT promotes TME remodeling by reducing immunosuppressive Tregs and M2 macrophages while enhancing CTL activity.

[0159] Local PAM-RT increases systemic T response and reduces Treg

[0160] To examine immune cells in secondary lymphoid organs, tumor-draining lymph nodes and spleen were harvested at 6 and 10 days after the start of RT, as in previous studies (e.g. Figure 10A ).like Figure 11A and 11B As observed, on days 6 and 10, the number of white blood cells in the spleen and draining lymph nodes of mice treated with PAM-RT was significantly increased. Figure 12A As observed, on days 6 and 10, the tumor draining lymph nodes (TDLN) of mice treated with PAM-RT showed significantly more CD8+ cells and a decreasing trend in Tregs. Spleen analysis revealed that, compared to a single ablation dose, in mice treated with PAM-RT, there was little change in CD8 T cells but a significant decrease in Tregs on day 6, which reversed by day 10, with significantly more CD8+ T cells and little change in Tregs. Figure 12B Studies of splenic T cell function using ELISPOT on days 6 and 10 revealed an increasing trend in granzyme B-secreting effector cells on day 6 in mice treated with PAM-RT. Figure 12C ), and the number of effector cells secreting IFNγ increased significantly ( Figure 12D Analysis on day 10 revealed a significant increase in effector cells secreting granzyme B and a trend toward an increase in effector cells secreting IFNγ. Although the treatments in these mice were limited to the primary tumor, the systemic modulation exhibited trends similar to those seen in tumors, namely decreased immunosuppression and enhanced T-cell responses.

[0161] Systemic PAM-RT delays metastasis progression and improves survival.

[0162] To investigate whether PAM-RT in metastatic organs could halt progression after low-fraction ablation RT of primary tumors in poorly immunogenic, highly metastatic cancers, local PAM-RT (22 Gy + 0.5 Gy x 4) was administered to orthotopic 4T1 breast cancer in Balb / c mice. Although PAM-RT of primary tumors tended to delay local tumor progression, survival was not improved, and all treated mice died from metastatic disease. Figures 13A to 13C Given that local treatment with PAM-RT reshapes the tumor microenvironment (TME), the use of PAM-RT was adapted to treat metastatic organs after primary tumor ablation. To achieve adequate local tumor control, BalB / C mice with palpable 4T1 tumors were treated with three doses of 20 Gy delivered to the primary tumor over three consecutive days. As expected, primary tumor ablation alone did not prevent lung metastases in the animals. Although an antitumor immune response was induced after primary tumor RT, RT-induced CTLs may not infiltrate metastatic sites. To test this, 12 days after primary tumor RT, the metastatic organ (i.e., the whole lung) was treated with a daily dose of 0.5 Gy for four days. Figure 14A Compared to primary tumor RT alone, these animals showed significantly improved survival rates after whole-lung PAM-RT, such as... Figure 14B As shown. Transfer load studies in treated mice revealed that, after injection of Indian ink, macroscopic examination (e.g.) revealed that... Figure 14C (as seen) and in histological specimens ( Figure 14D In studies, fewer metastatic lesions were found in the lungs treated with PAM-RT. PET scans of 4T1 mice showed similar results to lung acquisitions, with lungs receiving PAM-RT exhibiting a lower metastatic burden. Figure 15A and 15B These data suggest that a PAM-RT dose of 0.5 Gy x 4 can be directly applied to the primary tumor for local control, or that delayed PAM-RT can be applied to metastatic organs to slow tumor progression and improve survival when treating systemic diseases.

[0163] Systemic PAM-RT remodels metastatic niches and reduces lung Tregs

[0164] Characterization of immune cells in the lungs treated with PAM-RT showed results similar to those of local PAM-RT treatment, with a reduction in the immunosuppressive phenotype of these cells. Tregs were significantly reduced, such as... Figure 16A As observed, this led to a significant increase in the CD8 / Treg ratio in the entire lungs during PAM-RT treatment. Figure 26 ), Figure 16BThe image shows a whole-lung phenotypic analysis performed by flow cytometry 19 days after ablation of the primary tumor. Phenotypic analysis of T cells in the lungs revealed a significant increase in GzB secretion in both CD8+ and CD4+ T cells. Figure 16C Immunohistochemistry of micrometastases in a few metastatic lesions of the lung treated with PAM-RT showed a large infiltration of CD8+ T cells and a decrease in FoxP3+ cells compared to lesions in mice that received primary tumor ablation alone. Figure 16D In untreated animals, splenic metastasis was present, accompanied by splenomegaly and CD45+ leukopenia. Following primary tumor RT and primary tumor RT + lung PAM-RT, spleen size decreased, followed by CD45+ leukopenia. Figure 16E ) and an increase in the number of CD3+ T cells ( Figure 16F In the RT+lung PAM-RT treatment, lung macrophages recovered to baseline levels as seen in tumor-free wild-type animals. Peripheral myeloid dilatation was associated with G-CSF-secreting 4T1 tumors, and consistent with previous reports, splenic macrophages were significantly increased in CD45+ leukocytes in mice with 4T1 tumors, and these cells gradually decreased in mice treated with ablation alone compared to those treated with ablation+PAM-RT. Interestingly, in animals without treated tumors, splenic macrophages were immature and showed reduced class II MHC expression. The significant increase in class II MHC expression in macrophages after primary tumor ablation + / -lung PAM-RT treatment indicates that PAM-RT treatment activates macrophages (…). Figure 16G These experiments demonstrate that PAM-RT therapy can promote TME remodeling in both primary and metastatic tumors by reducing Tregs, activating macrophages to an inflammatory phenotype, and promoting the infiltration of CD8+CTLs in metastatic tumors.

[0165] Materials and methods

[0166] cell lines and mice

[0167] The mouse cell line Lewis lung cancer 3LL was purchased from ATCC and grown in supplemental DMEM (10% FBS, 5% sodium pyruvate, 2.5% NEAA, 1% antibacterial / antimitotic agent). The mouse breast cancer cell line 4T1 was purchased from ATCC and grown in supplemental DMEM (10% FBS, 1% antibacterial / antimitotic agent). Cell lines were used between passages 4 and 8, and mycoplasma was detected every 4 months using MycoAlert (Lonza LT07-705). Six- to eight-week-old C57BL / 6 mice and ten- to twelve-week-old BalB / C mice were ordered from the NCI, and athymic nude mice were ordered from Charles River. All studies conducted were approved by the Institutional Animal Care and Use Committee.

[0168] In vivo tumor research

[0169] C57BL / 6 mice were subcutaneously treated with 1.5 x 10 5 3LL cells were used to attack BalB / C mice in the mammary fat pad with 2x10 cells. 5 Four T1 cells were used for in situ attack. Tumors were allowed to grow to a diameter of 5 mm before treatment. Tumor size was measured twice weekly. Tumor volume was calculated using the following ellipsoidal formula: V = (π / 6 x length x width x height).

[0170] CT-guided radiotherapy in mice carrying tumors

[0171] Radiation was delivered using Xstrahl's Limited Small Animal Radiation Research Platform (SARRP). Image-guided radiation therapy was performed using SARRP's onboard cone-beam computed tomography (CBCT). After acquiring the CBCT, a treatment plan was constructed using Muriplan.

[0172] Tumor and immune cell analysis

[0173] Tumor cells were cultured, plated, and treated using the specified radiation protocol. Cells were harvested 6 and 24 hours after the final treatment on day 5. Cells were collected and stained for flow cytometry. Bone marrow-derived macrophages were polarized to M1 (100 ng / mL LPS + 50 ng / mL IFNg) and M2 (10 ng / mL IL-4) and treated with radiation 24 hours later. T cell populations were sorted from the spleens of naive C57BL / 6 mice using CD3, CD8, CD4, and CD25 antibodies and incubated overnight before radiation treatment. Immune cells were harvested 6 hours after the final radiation dose.

[0174] Tumor processing

[0175] Tumors or whole lungs were harvested on ice, weighed, and washed. After manual dissection with a blade, the tumors or whole lungs were transferred to 1 mL of digestion buffer (10% FBS, 100 u / mL collagenase I and IV (Sigma), and 1x DNase I (Thermo Scientific)) in 15 mL conical tubes with magnetic stirrers. The tubes were incubated at 37°C with rotation for 15 minutes, then transferred to a stirring plate for manual digestion for 15 minutes. The single-cell suspension was filtered. The cells were resuspended for flow cytometry.

[0176] Flow cytometry analysis

[0177] Cells were stained with surface staining antibodies at 4°C for 30 minutes. The antibodies used included CD45, CD3, CD4, FOXP3, CD8, GzB, CD11b, MHCII, IL-10, TNFα, and CD206. After washing, cells were fixed or permeabilized with 4% PFA for intracellular staining, using BD Pharminogen transcription factor buffer as per manufacturer's instructions. Intracellular staining agents included FOXP3, TNFα, IL-10, and granzyme B, followed by fixation with 4% PFA. Cells were collected using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).

[0178] Lymphoid organ collection for immunoassay

[0179] Spleens and draining lymph nodes were harvested on ice and processed into single-cell suspensions. In the spleen, erythrocytes were lysed using ACK lysis buffer (Lonza). Cells were counted and then resuspended in complete RPMI (10% heat-inactivated FBS, 1% antibiotic / antimitotic agent). For intracellular cytokine analysis, Golgi-stop and monensin were added at 37°C for 3 hours. The single-cell suspensions were further processed for flow cytometry analysis, as performed on tumors.

[0180] ELISOPT Measurement

[0181] Spleen cells prepared for immunoassay were seeded at 1 x 10^6 cells per well on coated ELISPOT plates and incubated overnight before further processing. Reagents for IFNγ ELISPOT were ordered from BD Biosciences and followed the manufacturer's instructions. Reagents for granzyme B ELISPOT were ordered from R&D and followed the manufacturer's instructions.

[0182] Histological staining of tumor

[0183] Tumors and lungs were collected and transferred to 4% PFA or 10% formalin and stored at 4°C. The samples were then transferred to 70% PFA and embedded in paraffin. The blocks were sectioned and double-stained with CD8 and FOXP3.

[0184] Statistical analysis

[0185] Statistical analysis was performed using PRISM 7 (Graphpad Software). Student's t-test or ANOVA with multiple comparisons was used. Data are presented as mean ± standard deviation. Survival curves were analyzed using the Mantel-Cox log-rank test and the Grehan-Breslow-Wilcoxon test. For statistical significance, p-values ​​are expressed as *p<0.05, **p<0.005, ***p<0.0005, and ****p<0.0001.

[0186] RT-PCR analysis of TME

[0187] Six days after treatment initiation, tumors were harvested and weighed on ice. The tumors were then manually dissected on ice and subsequently transferred to RNA. For processing, samples were thawed on ice, and RNA was extracted using Trizol according to the manufacturer's protocol. RNA was quantified on Nanodrop at a 260nm / 280nm ratio of approximately 2. cDNA synthesis was performed using the Verso cDNA Synthesis Kit (Thermo Fisher) according to the kit protocol. RT-PCR was performed in 384-well plates on an ABI 7900HT using the Powerup SYBR Green Kit (Thermo Fisher). Primers used were FOXP3 F-ACTCGCATGTTCGCCTACTTCAG and R-GGCGGATGGCATTCTTCCAGGT, and β2-microglobulin F-TTCTGGTGCTTGTCTCACTGA and R-CAGTATGTTCGGCTTCCCATTC. Delta CT was calculated using SDS2.4 and compared with an untreated control.

[0188] PET imaging

[0189] Mice were fasted overnight and brought to the imaging facility. 300–400 uCi (12–15 MBq) of [18F]fluoro-2-deoxyglucose (FDG) in 0.1 ml of physiological saline was injected via the tail vein, and imaging began 1 hour post-injection. Mice were anesthetized with isoflurane and imaged on an Inveon Multimodality scanner (Siemens). Analysis was performed using ASIPRO and IRW (both from Siemens) proprietary software.

[0190] Lungs treated with Indian ink

[0191] Mice carrying 4T1 tumors were euthanized after treatment. Lungs were inflated by intratracheal injection of 10% Indian ink. Lungs were harvested and washed in 1 L of water, then stored in Feket solution (300 ml 70% EtOH, 30 ml 37% formaldehyde, 5 ml glacial acetic acid) to bleach large metastases. Metastatic lesions were counted.

[0192] Example 2: Quantitative and qualitative T-cell analysis after radiotherapy

[0193] To test the radiation therapy protocol, six-week-old C57BL / 6 mice were subcutaneously injected with 3LL on the dorsum of their paws. Tumors were allowed to grow to 50 mm before treatment. 3 Tumor size was measured three times per week during the study. Tumor volume was calculated using the following ellipsoidal formula: V = (π / 6 x length x width x height).

[0194] The inoculated mice were randomly divided into four treatment groups. Group 1 received no treatment; Group 2 received a single 24 Gy dose; Group 3 received a single 22 Gy dose followed by four 0.5 Gy doses (administered one day apart) one day later; and Group 4 received four 0.5 Gy doses (administered one day apart) followed by a single 22 Gy dose one day later. Tumor size was measured three times weekly, and survival was assessed daily. Figure 18A As shown, mice in group three (c) had better survival outcomes than mice in groups one, two, and four. Several mice in the third treatment group survived for nearly three months, while all mice in groups one and two, and most mice in group four, died after approximately one month. Another group of mice was treated as above and injected with FITC-glucan six days after the first treatment to quantify the tumor vascular system. Figure 18B As shown, compared with the first and second groups of mice, the third and fourth groups of mice exhibited increased vascular density.

[0195] To investigate potential immune effects, additional experiments were conducted using immunocompromised C57Bl6 mice and nude mice. These mice were divided into three groups and treated as described in the first, second, and third groups above. In the C57Bl6 mice, tumor growth in the third group was slower than in the first and second groups. Figure 19A As observed. This effect disappears in nude mice, such as... Figure 19B As shown. Similarly, compare Figure 20A and 20B Consequently, the survival benefit observed in C57Bl6 mice was not observed in nude mice.

[0196] To investigate the effect of radiotherapy on the immune response, three different tumor cell lines were used: 1x10 5 3LL, 2x10 5 UN-KC-6141 and 4x10 5 MOE HPV E6 / 7 / H-Ras cells were used. C57Bl6 mice were inoculated with the above cells, and mice treated with each cell type were divided into six treatment groups: (1) no treatment, (2) 24 Gy, (3) 20 Gy followed by 1 Gy for four daily doses after 1 day, (4) 22 Gy followed by 0.5 Gy for four daily doses after 1 day, (5) 1 Gy for four daily doses, and (6) 0.5 Gy for four daily doses. Six days after the first treatment, tumors were collected, and leukocyte infiltration was analyzed by flow cytometry as described below. For all cell lines tested, increased leukocyte infiltration was observed in group 3 compared with the other groups, see [link to relevant documentation]. Figures 21A to 21C In mice treated with H-Ras cells, group 4 was more effective than groups 1, 2, 5, and 6. Figure 21B However, this result was not observed in mice treated with 3LL or 4T1 tumor cells.

[0197] To determine the effectiveness of the above radiation regimens in combination therapy, mice were injected with the aforementioned tumor cells and treated with: trabectadin alone; 24 Gy and trabectadin; or 22 Gy followed by three weekly doses of 0.5 Gy and three weekly doses of trabectadin one day later. Survival and tumor growth were recorded for each group. Figure 22A The survival rates of mice in different groups were shown, with the highest survival rates observed in the groups treated with 22 Gy and subsequently with four daily doses of 0.5 Gy and trabectedine after 1 day. Figures 22B to 22D Growth curves for different tumors within each treatment group are shown, with the slowest growth rate observed in the group receiving 22 Gy followed by four daily doses of 0.5 Gy and trabectedine after one day. Figure 22D ).

[0198] Further combination experiments were conducted using mice inoculated with 4T1 cells into the fourth mammary fat pad. Approximately eight days later, the mice were treated as follows:

[0199] 1. Three doses of 20 Gy,

[0200] 2. Three doses of 20 Gy and anti-PD1 therapy,

[0201] 3. Three doses of 20 Gy followed by four daily doses of 0.5 Gy (administered to the whole lung) after 12 days, or

[0202] 4. Three doses of 20 Gy and anti-PD1 therapy, followed by four daily doses of 0.5 Gy (administered to the whole lung) and anti-PD1 therapy after 12 days.

[0203] Treatment plan shown Figure 23 In the middle. For example Figure 24A and 24B As shown (excluding mice whose survival might be confounded by local failure of the primary tumor), mice treated in group 4 exhibited the longest survival. Mice administered 4T1 cells intravenously and treated with the following regimens had similar survival rates to untreated mice: anti-PD1 therapy alone, 0.5 Gy at 4 daily doses alone, or anti-PD1 therapy and 0.5 Gy at 4 daily doses, see [link to relevant documentation]. Figure 25 .

[0204] method

[0205] cell lines and mice

[0206] The mouse cell line Lewis lung cancer 3LL was purchased from ATCC and grown in supplemented DMEM (10% FBS, 5% sodium pyruvate, 2.5% NEAA, 1% Pen / Strep). The 4T1 mouse breast cancer cell line was purchased from ATCC and grown in supplemented DMEM (10% FBS, 1% Pen / Strep).

[0207] Six-week-old C57BL / 6 mice were ordered from the NCI and maintained under pathogen-free conditions.

[0208] CT-guided radiotherapy in mice carrying tumors

[0209] Radiation was delivered using Xstrahl's Limited Small Animal Radiation Research Platform (SARRP). Image-guided radiation therapy was performed using SARRP's onboard cone-beam computed tomography (CBCT). After acquiring the CBCT, a treatment plan was constructed using Muriplan.

[0210] Immunotherapy in mice carrying tumors

[0211] With or without antigen vaccination, tumor-carrying mice randomly assigned to the immunotherapy treatment group were given 200 μg PD-1 i.p. (every three days for a total of 5 doses), αCD40, 4-1BBL, or GM-CSF.

[0212] Syntac application

[0213] A Syntac construct conjugated with the T cell co-stimulatory domain 41BBL to a tumor-specific antigen will be used to induce activated antigen-specific effector CD8+ T cells. Tumor-carrying mice randomly assigned to Syntac received a single 300 μg per ip treatment. Mice treated with antigen-specific Syntac were compared to mice treated with either unrelated Syntac or the co-stimulatory domain 4-1BBL alone.

[0214] Tumor digestion

[0215] Tumors were collected and weighed on ice. After being manually dissected into 1 mm x 1 mm pieces with a blade, the tumors were transferred to 1 mL of digestion buffer (5% FBS, 100 u / mL collagenase I and IV (Sigma), and 1x DNase I (Thermo Scientific)) in a 15 mL conical tube with a magnetic stir bar. The tubes were incubated at 37°C for 30 minutes and then transferred to a stirring plate for manual digestion for 30 minutes. The single-cell suspension was filtered and resuspended for analysis.

[0216] Flow cytometry analysis of immune cells

[0217] Single-cell suspensions were stained with surface staining antibodies at 4°C for 30 minutes. After washing, cells were fixed or permeabilized with 4% PFA for intracellular staining. BD Pharmingen transcription factor buffer was prepared according to the manufacturer's instructions. Intracellular staining agents were FOXP3 and Ki-67, followed by fixation with 4% PFA. Cells were collected on an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).

[0218] Antibody

[0219] Immunofluorescence antibodies: The primary antibodies used were CD3, CD8, CD4, CD11b, and Gr1 (BD Pharminogen). The secondary antibodies were goat anti-rabbit FITC and goat anti-rat APC (Abcam).

[0220] Flow cytometry antibodies: CD45, CD3, CD4, CD8, CD69, PD1, CD11b, Gr1, Ly6C, LY6G, CD11c, NK1.1, NKG2D, MHC II(IA) and F4 / 80.

[0221] Tumor microenvironment analysis

[0222] Six days after treatment, tumors were harvested and weighed on ice. While remaining on ice, the tumors were transferred to 1.7 mL Eppendorf tubes, and each milligram of tissue was homogenized in 4.5 μL PBS containing 1X protease and a phosphatase inhibitor (Cell signaling). The tubes were centrifuged at 14,000 g for 15 minutes. The supernatant was transferred to clean tubes and stored at -80°C.

[0223] Vascular density quantification using FITC-glucan

[0224] Mice were subcutaneously injected with 1 x 10^5 3 mL tumor cells at the flank. Tumor growth was allowed for 14 days to approximately 6 mm. Mice were randomly assigned to receive no treatment, 24 Gy on day 1, 22 Gy + 0.5 Gy x 4, or 0.5 Gy x 4 starting from day 2. Six days after treatment initiation, 70 kDa FITC dextran (25 mg / mL) was injected intravenously and circulated for 20 minutes. Mice were then sacrificed, and blood and tumors were collected. Tumors were weighed and incubated overnight at 37°C with 1.5 mL of collagenase IV. Tumors were then homogenized, and cell debris was centrifuged at 14,000 rpm for 5 minutes. The supernatant was collected and fluorescence detected at 485 / 535. Fluorescence per milligram was then calculated. Serum was used as an injection control.

[0225] While preferred embodiments of the invention have been shown and described herein, it will be readily understood by those skilled in the art that such embodiments are provided by way of example only. Many modifications, alterations, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in the practice of the invention. The following claims are intended to define the scope of the invention and therefore cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A computer-readable medium configured with instructions that, when executed, cause a processor to: provide instructions to a radiotherapy system to deliver an ablation dose of radiotherapy to a first region, and then deliver a subablation dose to a second region after the ablation dose, wherein the subablation dose is applied after the ablation dose, and wherein the ablation dose at the first region is between 20 and 100 Gy.

2. The computer-readable medium of claim 1, wherein the subablation dose is delivered at least 1 hour after the ablation dose.

3. The computer-readable medium of claim 1, wherein the subablation dose is delivered at least 1 day after the ablation dose.

4. The computer-readable medium of claim 1, wherein the subablation dose is delivered no more than 4 days after the ablation dose.

5. The computer-readable medium of claim 1, wherein the cumulative amount of radiotherapy delivered to the second region throughout the treatment includes an amount of radiotherapy less than the ablation dose.

6. The computer-readable medium of claim 5, wherein the cumulative amount of radiotherapy less than the ablation dose comprises a plurality of subablation doses.

7. The computer-readable medium of claim 1, wherein the first region includes the region of the tumor.

8. The computer-readable medium of claim 7, wherein the second region includes the region of the tumor.

9. The computer-readable medium of claim 1, wherein the first region includes the region of the first tumor and the second region includes the region of the second tumor.

10. The computer-readable medium of claim 9, wherein the first tumor comprises a primary tumor and the second tumor comprises a metastatic tumor.

11. The computer-readable medium of claim 9, wherein the first tumor comprises a metastatic tumor and the second tumor comprises a primary tumor.

12. The computer-readable medium of claim 9, wherein the second region comprises a plurality of second regions, and each of the plurality of second regions receives a cumulative amount of radiation less than the ablation dose.

13. The computer-readable medium of claim 1, wherein the second region includes a region different from the first region.

14. The computer-readable medium of claim 1, wherein the second region includes the region of the tumor.

15. The computer-readable medium of claim 1, wherein the second region includes a region that may develop into a metastatic tumor.

16. The computer-readable medium of claim 1, wherein the second region comprises a region of an organ selected from the group consisting of: bones, lymph nodes, lungs, liver, brain, adrenal glands, breasts, eyes, kidneys, muscles, pancreas, salivary glands, and spleen.

17. The computer-readable medium of claim 1, wherein the second region comprises the entire body of the object scanned with the subablation dose.

18. The computer-readable medium of claim 1, wherein the first region comprises a region of primary tumors of organs selected from the group consisting of: breast, bladder, brain, colon, rectum, endometrium, kidney, pancreas, prostate, liver, lung, skin, thyroid, uterus, lymph nodes, tonsils, thymus, spleen, and bone marrow, and the second region comprises a region of metastatic tumors of organs selected from the group consisting of: bone, lymph nodes, lung, liver, brain, adrenal gland, breast, eye, kidney, muscle, pancreas, salivary gland, and spleen.

19. The computer-readable medium of claim 1, wherein the first region comprises a region of metastatic tumors of organs selected from the group consisting of: bone, lymph nodes, lung, liver, brain, adrenal gland, breast, eye, kidney, muscle, pancreas, salivary gland, and spleen, and the second region comprises a region of primary tumors of organs selected from the group consisting of: breast, bladder, brain, colon, rectum, endometrium, kidney, pancreas, prostate, liver, lung, skin, thyroid gland, uterus, lymph nodes, tonsils, thymus, spleen, and bone marrow.

20. The computer-readable medium of claim 1, wherein the first region includes an identified tumor and the second region does not include an identified tumor.

21. A radiotherapy system comprising: Provides radioactive sources with ablation dose and subablation dose; A processor coupled to the radiation source; as well as The computer-readable medium as claimed in claim 1, The processor is configured with instructions from the computer-readable medium.

22. The radiotherapy system of claim 21, wherein the ablation dose at the first region is between 20 and 60 Gy.

23. The radiotherapy system of claim 21, wherein the subablation dose is between 0.1 and 2 Gy.

24. The radiotherapy system of claim 21, wherein the subablation dose comprises a plurality of subablation doses and each of the plurality of subablation doses at the second region is between 0.1 and 2 Gy.

25. The radiotherapy system of claim 21, wherein the subablation dose is between 0.1 and 0.5 Gy.

26. The radiotherapy system of claim 21, wherein the subablation dose comprises a plurality of subablation doses and each of the plurality of subablation doses at the second region is between 0.1 and 5 Gy.

27. The radiotherapy system of claim 21, wherein three subablation doses are administered.

28. The radiotherapy system of claim 21, wherein more than three subablation doses are administered.

29. The radiotherapy system of claim 21, wherein the first subablation dose is administered within 24 hours after the ablation dose is administered.

30. The radiotherapy system of claim 21, wherein the first subablation dose is administered between 6 and 26 hours after the ablation dose is applied.

31. The radiotherapy system of claim 21, wherein the application of the ablation dose and the subablation dose reduces the size or intensity of the treated tumor, the size or intensity being measured by imaging selected from the group consisting of: computed tomography, magnetic resonance imaging, and positron emission tomography.

32. The radiotherapy system of claim 21, wherein the administration of the ablation dose and the subablation dose improves the survival rate of the subject, reduces the number or severity of symptoms experienced by the subject, increases the number of immune cells in the tumor microenvironment, or increases the number of activated immune cells in the tumor microenvironment.

33. The radiotherapy system of claim 21, wherein the radiation is selected from the group consisting of: X-ray radiation, gamma-ray radiation, alpha particle radiation, beta particle radiation, neutron particle radiation, external beam radiation, and brachytherapy.

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