Post-radiation therapy ablation modulation

Abstract

To provide a method and a system for treating a cancer in a subject.SOLUTION: A method includes providing an ablative dose of radiation therapy to a first region including a cancer followed by a sub-ablative dose to a second region, wherein the sub-ablative dose is administered within 1 hour to 4 days after the ablative dose.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 892,273, filed August 27, 2019, which is incorporated herein by reference in its entirety.

[0002] Federally Sponsored Research Statement This invention was made with government support under R01CA22686(CG), 1S10OD019961-01 and 1S10RR029545-01 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] Cancer is a leading cause of death worldwide and is currently the second leading cause of death in the United States. Traditional treatment options for tumors include cytotoxic therapies such as radiation therapy (RT) and chemotherapy. Local tumor control by surgery or RT, even in the presence of systemic therapy, can ultimately fail due to metastatic progression. More recently, immunotherapy, both alone and in combination with other therapies, has been proposed as a viable treatment option for inhibiting metastatic progression. Immunotherapies include checkpoint inhibitors, tumor vaccines, and adoptive cell transfer. While immunotherapy has shown promise in the treatment of several solid tumors, such as melanoma and renal cell carcinoma, immunotherapy has been less successful in other, more fibrous tumors, such as pancreatic cancer. Even within cancer types, there are subsets of responders and non-responders, resulting in a large number of individuals being treated less than ideally.

[0004] Although RT may be used to treat cancer, its cytotoxic effects may limit its usefulness. For example, RT administered to a tumor located in an organ may substantially damage the organ in which the tumor is located. A metastatic tumor may be located at a distance from the primary tumor, resulting in the primary tumor and metastatic tumor being located in more than one organ. In at least some instances, RT administered to more than one organ may damage more than one organ due to its cytotoxic effects.

[0005] In view of the above, it would be desirable to have improved methods and devices for treating cancer, ideally capable of treating tumors located in delicate organs and tissue structures while inhibiting the metastatic progression of cancer and causing damage. Summary of the Invention

[0006] The present methods and devices provide improved treatment for cancer using RT. Tumors can be treated with an ablative dose of radiation followed by a sub-ablative treatment at the same or a different location. The subsequent sub-ablative treatment may be administered within about one hour to about four days after the ablative treatment to induce an adaptive immune response to cancer cell antigens. The subsequent sub-ablative treatment may be administered at multiple locations, including the same tumor location as the ablative dose, a different tumor, or a location prone to metastasis. In some embodiments, the time between the ablative and sub-ablative treatments allows for structural changes in the tumor microenvironment, including the tumor vasculature. Because the subsequent treatment is sub-ablative, the subsequent treatment may be administered at multiple locations, even in locations without a defined tumor. The subsequent treatment may also include, for example, systemic treatment. Radiation therapy may be administered in a number of ways, including using a radiotherapy device or brachytherapy, and combinations thereof.

[0007] While treatment may be performed in a number of ways, in some embodiments, an initial ablative radiation therapy treatment generates a significant number of antigens, which are presented to the immune system. An adaptive immune response is generated against the antigens presented in the initial treatment. Subsequent sub-ablative treatments can affect the tumor microenvironment, increasing the extent to which immune cells can enter the tumor. Subsequent sub-ablative treatments can result in increased tumor perfusion (e.g., "cracking" the tumor) that alters the tumor microenvironment and can result in other changes to the tumor microenvironment that promote an immune response. While reference is made to treating tumors with sub-ablative doses of RT, RT can also be used prophylactically to treat tumor-prone tissues to inhibit tumor growth in those tissues.

[0008] In some embodiments, described herein are methods of treating cancer in a subject, the methods comprising delivering an ablation dose of radiation therapy to a first region containing the cancer, followed by a sub-ablative dose to a second region, wherein the sub-ablative dose is administered after the ablation dose. In some embodiments, the sub-ablative dose is administered at least one hour after the ablation dose. In some embodiments, the sub-ablative dose is administered at least one day after the ablation dose. In some embodiments, the sub-ablative dose is administered at most four days after the ablation dose.

[0009] In some embodiments, disclosed herein are methods of treating cancer in a subject, the method comprising delivering an ablation dose of radiation therapy to a first region comprising the cancer, followed by a sub-ablative dose to a second region, wherein the sub-ablative dose is administered within 1 hour to 4 days after the ablation dose. In some embodiments, the cumulative amount of radiation therapy delivered to the second region over the course of treatment comprises a sub-ablative dose. In some embodiments, the cumulative sub-ablative dose comprises multiple sub-ablative doses. In some embodiments, the first region comprises a region of a tumor, and optionally the second region comprises a region of a tumor. In some embodiments, the first region comprises a region of a first tumor, and the second region comprises a region of a second tumor. In some embodiments, the first tumor comprises a primary tumor and the second tumor comprises a metastatic tumor. In some embodiments, the first tumor comprises a metastatic tumor and the second tumor comprises a primary tumor. In some embodiments, the second region includes a plurality of second regions, each of which receives a cumulative dose of radiation therapy that is 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 region of a tumor. In some embodiments, the second region includes a region prone to developing metastatic tumors, and optionally, the second region includes a region of an organ selected from the group consisting of bone, lymph node, lung, liver, brain, adrenal gland, breast, eye, kidney, muscle, pancreas, salivary gland, and spleen. In some embodiments, the second region includes the entire body of the subject scanned using a sub-ablative dose. In some embodiments, the first region comprises a region of a primary tumor in an organ selected from the group consisting of breast, bladder, brain, colon, rectum, endometrium, kidney, pancreas, prostate, liver, lung, skin, thyroid, uterus, lymph node, tonsil, thymus, spleen, and bone marrow, and the second region comprises a region of a metastatic tumor in an organ selected from the group consisting of bone, lymph node, lung, liver, brain, adrenal gland, breast, eye, kidney, muscle, pancreas, salivary gland, and spleen.In some embodiments, the first region comprises a region of a metastatic tumor in an organ selected from the group consisting of bone, lymph node, lung, liver, brain, adrenal gland, breast, eye, kidney, muscle, pancreas, salivary gland, and spleen, and the second region comprises a primary tumor in an organ selected from the group consisting of breast, bladder, brain, colon, rectum, endometrium, kidney, pancreas, prostate, liver, lung, skin, thyroid, uterus, lymph node, tonsil, thymus, spleen, and bone marrow. In some embodiments, the first region comprises an identified tumor and the second region does not comprise an identified tumor.

[0010] In some embodiments, disclosed herein is a computer-readable medium configured with instructions that, when executed, cause a processor to provide instructions to a radiation therapy system to deliver an ablation dose of radiation therapy to a first region and deliver a sub-ablative dose to a second region following the ablation dose. In some embodiments, the sub-ablative dose is delivered at least one hour after the ablation dose. In some embodiments, the sub-ablative dose is delivered at least one day after the ablation dose. In some embodiments, the sub-ablative dose is delivered at most four days after the ablation dose.

[0011] In some embodiments, disclosed herein is a computer-readable medium configured with instructions that, when executed, cause a processor to provide instructions to a radiation therapy system for an ablation dose of radiation therapy to a first region, followed by a sub-ablative dose to a second region within 1 hour to 4 days after the ablation dose. In some embodiments, the cumulative amount of radiation therapy provided to the second region over the course of treatment comprises an amount of radiation therapy that is less than the ablation dose. In some embodiments, the cumulative amount of radiation therapy that is less than the ablation dose comprises multiple sub-ablative doses. In some embodiments, the first region comprises a region of a tumor, and optionally the second region comprises a region of a tumor. In some embodiments, the first region comprises a region of a first tumor, and the second region comprises a region of a second tumor. In some embodiments, the first tumor comprises a primary tumor and the second tumor comprises a metastatic tumor. In some embodiments, the first tumor comprises a metastatic tumor and the second tumor comprises a primary tumor. In some embodiments, the second region includes a plurality of second regions, each of which receives a cumulative dose of radiation therapy that is 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 region of a tumor. In some embodiments, the second region includes a region prone to developing metastatic tumors, and optionally, the second region includes a region of an organ selected from the group consisting of bone, lymph node, lung, liver, brain, adrenal gland, breast, eye, kidney, muscle, pancreas, salivary gland, and spleen. In some embodiments, the second region includes the entire body of the subject scanned using a sub-ablative dose. In some embodiments, the first region comprises a region of a primary tumor in an organ selected from the group consisting of breast, bladder, brain, colon, rectum, endometrium, kidney, pancreas, prostate, liver, lung, skin, thyroid, uterus, lymph node, tonsil, thymus, spleen, and bone marrow, and the second region comprises a region of a metastatic tumor in an organ selected from the group consisting of bone, lymph node, lung, liver, brain, adrenal gland, breast, eye, kidney, muscle, pancreas, salivary gland, and spleen.In some embodiments, the first region comprises a region of a metastatic tumor in an organ selected from the group consisting of bone, lymph node, lung, liver, brain, adrenal gland, breast, eye, kidney, muscle, pancreas, salivary gland, and spleen, and the second region comprises a primary tumor in an organ selected from the group consisting of breast, bladder, brain, colon, rectum, endometrium, kidney, pancreas, prostate, liver, lung, skin, thyroid, uterus, lymph node, tonsil, thymus, spleen, and bone marrow. In some embodiments, the first region comprises an identified tumor and the second region does not comprise an identified tumor.

[0012] In some embodiments, disclosed herein is a radiation therapy system, the radiation therapy system including a radiation source providing an ablation dose and sub-ablative doses, and a processor coupled to the radiation source, the processor configured with the instructions described above. In some embodiments, the ablation dose comprises between 20 Gy and 100 Gy in a first region. In some embodiments, the ablation dose comprises between 20 Gy and 60 Gy in a first region. In some embodiments, the sub-ablative dose comprises between 0.1 Gy and 2 Gy, optionally the sub-ablative dose comprises multiple sub-ablative doses, each of the multiple sub-ablative doses comprising between 0.1 Gy and 2 Gy in a second region. In some embodiments, the sub-ablative dose comprises between 0.1 Gy and 0.5 Gy, optionally the sub-ablative dose comprises multiple sub-ablative doses, each of the multiple sub-ablative doses comprising between 0.1 Gy and 5 Gy in the second region. In some embodiments, three sub-ablative doses are administered. In some embodiments, more than three sub-ablative doses are administered. In some embodiments, the first sub-ablative dose is administered within 24 hours after administration of the ablative dose. In some embodiments, the first sub-ablative dose is administered between 6 and 26 hours after administration of the ablative dose. In some embodiments, the treatment reduces the size or intensity of the treated tumor as measured by an imaging method selected from the group consisting of computed tomography scan, magnetic resonance imaging, positron emission tomography, and computed tomography scan. In some embodiments, the treatment increases the subject's survival rate, reduces the number or severity of symptoms the subject develops, 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 irradiation, gamma irradiation, alpha particle irradiation, beta particle irradiation, neutron particle irradiation, external beam radiation, and brachytherapy.

[0013] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0014] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth embodiments in which the principles of the invention are utilized, and the accompanying drawings in which: [Figure 1] 1 illustrates a radiation therapy system suitable for use with methods and programs according to some embodiments of the present disclosure. [Figure 2A] According to some embodiments of the present disclosure, an example of a tumor in a first region is a subject having a tumor in the liver. [Figure 2B] 1 illustrates a subject being treated in two regions, including a first region containing a first tumor and a second region containing a second tumor, according to some embodiments of the present disclosure. [Figure 3] 1 illustrates examples of first, second, and third treatment areas of a subject, where the first treatment area includes a tumor in the lung, the second treatment area includes the entire lung, and the third treatment area includes a metastatic tumor, according to some embodiments of the present disclosure. [Figure 4] 1 illustrates a process for treating a patient according to some embodiments of the present disclosure. [Figure 5A] 5A and 5B illustrate two models of radio-priming according to some embodiments of the present disclosure: Figure 5A illustrates a pre-priming method in which four sub-ablative doses are administered to the tumor to stimulate the immune system before the ablative dose is administered. [Figure 5B]5A and 5B illustrate two models of radio-priming according to some embodiments of the present disclosure. Figure 5B illustrates a post-ablative conditioning therapy method in which an ablative dose of radiation therapy is administered to the tumor, followed by four sub-ablative doses, with the first ablative dose activating effector T cells and the later sub-ablative doses altering the tumor microenvironment to increase immune infiltration, alter the cytokine milieu, and reprogram macrophages. [Figure 6] 1 illustrates a computer system for use in accordance with some embodiments of the present disclosure. [Figure 7A] The treatment scheme is shown in Figure 7A-D, comparing tumor growth before and after priming with a control group of 3LL tumor-bearing mice. Log-rank (Mantel-Cox) analysis revealed * indicates p<0.05. n=5 in Figures 7A-D. [Figure 7B] Relative tumor growth of treatment groups is depicted. [Figure 7C] The initial tumor volume tripling time is illustrated. [Figure 7D] Survival rates of treated mice are depicted. [Figure 8A] A schematic treatment scheme for local PAM and treatment groups are shown. n = 4–5 mice. [Figure 8B] As in Figure 8A, relative tumor growth curves for treated mice are shown, n = 4-5 mice. [Figure 8C] Initial tumor volume tripling times are shown for treated 3LL tumor-bearing mice. n = 28-35 mice. [Figure 8D] Figure 1 shows combined survival curves across multiple experiments in 3LL tumor-bearing mice. * indicates p<0.05 by log-rank (Mantel-Cox) and Gehan-Wilcoxon-Breslow tests. [Figure 8E] This figure illustrates tumor growth in nude mice bearing 3LL tumors. n = 10-13 mice. [Figure 8F]Survival rate of 3LL tumor-bearing nude mice. n = 10-13 mice. [Figure 9A] 9A-9C relate to in vitro treatment of 3LL tumor cells. Figure 9A illustrates an outline of the in vitro treatment scheme of PAM of 3LL tumor cells. [Figure 9B] Figures 9A-9C relate to in vitro treatment of 3LL tumor cells. Figure 9B illustrates cell death as measured by LIVE / DEAD Fixable Dye 6 and 24 hours after treatment as in Figure 9A. [Figure 9C] Figures 9A-9C relate to in vitro treatment of 3LL tumor cells. Figure 9C illustrates phenotypic immunomodulatory, stress, and immunosuppressive markers by surface expression 6 and 12 hours after the final treatment as in Figure 9A. *p<0.05, **p<0.005, ***p<0.0005, and ****p<0.0001 as determined by t-test. [Figure 9D] Figures 9D-G relate to in vitro treatment of subsets of immune populations with 0.5 Gy x 4. Figure 9D illustrates the viability of sorted T cell subsets after treatment. [Figure 9E] Figures 9D-G relate to in vitro treatment of a subset of immune populations with 0.5 Gy x 4. Figure 9E illustrates CD25 and FOXP3 expression after treatment of selected CD4+CD25+FOXP3+ (Tregs). [Figure 9F] Figures 9D-G relate to in vitro treatment of a subset of immune populations with 0.5 Gy x 4. Figure 9F illustrates CD206 expression, an M2 macrophage marker, on cytokine-polarized bone marrow-derived macrophages. [Figure 9G] Figures 9D-G relate to in vitro treatment of a subset of immune populations with 0.5 Gy x 4. Figure 9G illustrates cytokine secretion of cytokine-polarized bone marrow-derived macrophages after treatment. *p<0.05, **p<0.005, ***p<0.0005, and ****p<0.0001 as determined by t-test. [Figure 10A]Schematic diagram of treatment of 3LL tumor-bearing mice and harvest for localized PAM treatment. [Figure 10B] Figure 1 illustrates in vivo infiltrating leukocytes and Tregs by flow cytometry at days 6 and 10 after initiation of topical PAM treatment. *p<0.05, **p<0.005 by ANOVA multiple comparisons. [Figure 10C] Figure 1 depicts tumor lysate total RNA expression of FOXP3 on day 6. *p<0.05, **p<0.005 by ANOVA multiple comparisons. [Figure 10D] Illustrates intratumoral infiltration of granzyme B-secreting effector cells on days 6 and 10 by flow cytometry. [Figure 10E] Flow cytometry analysis of tumor-infiltrating macrophage polarization phenotypes at days 6 and 10 after treatment initiation is shown. *p<0.05, **p<0.005 by ANOVA multiple comparisons. [Figure 11A] Flow cytometry shows the percentage of lymphocytes in the spleen on days 6 and 10. *p<0.05, **p<0.005 in all panels by ANOVA multiple comparisons. [Figure 11B] Flow cytometry shows the percentage of lymphocytes in the draining lymph nodes on days 6 and 10. *p<0.05, **p<0.005 in all panels by ANOVA multiple comparisons. [Figure 12A] Figure 1 illustrates the characteristics of CD8 T cell and Treg populations in draining lymph nodes at days 6 and 10 after local PAM treatment. *p<0.05, **p<0.005 by t-test, n=4-6. [Figure 12B] Figure 1 illustrates the characteristics of CD8 T cell and Treg populations in the spleen at days 6 and 10 after local PAM treatment. *p<0.05, **p<0.005 by t-test, n=4-6. [Figure 12C]ELISPOT for granzyme B of polyfunctional cytokine secretion in spleens of treated mice on days 6 and 10 is shown. *p<0.05 by t-test, n=4-6. [Figure 12D] ELISPOT for IFNγ of polycytokine secretion in spleens of treated mice on days 6 and 10 is shown. *p<0.05 by t-test, n=4-6. [Figure 13A] Tumor measurements are shown for orthotopic 4T1 tumor-bearing mice treated with topical PAM and control groups. n = 12–13 mice. [Figure 13B] Relative tumor growth in 4T1 tumor-bearing mice using local PAM is shown. n = 12–13 mice. [Figure 13C] Figure 1 shows the survival curves of 4T1 mice treated with topical PAM. n = 12–13 mice. [Figure 14A] 1 illustrates a schematic of systemic treatment of PAM with whole-lung irradiation following ablation of the primary tumor. [Figure 14B] Figure 1 shows the overall survival and survival 2 months after inoculation following systemic PAM treatment compared to primary tumor ablation alone. * indicates p<0.05 by log-rank (Mantel-Cox) and Gehan-Wilcoxon-Breslow tests. n=26–27 mice. [Figure 14C] Graph of India ink injected lungs on day 12 with and without whole lung irradiation (red arrows indicate macrometastases) with enumerated visible macrometastases. [Figure 14D] Histological sections of lungs (red asterisks indicate metastatic foci) are shown, along with a graph of enumerated foci. [Figure 15A] PET scan 28 days after ablation of the primary tumor is shown, representative of seven images. [Figure 15B] PET scans 28 days after ablation of the primary tumor and 12 days after whole-lung irradiation are shown, representative of six images. [Figure 16A] Figure 1 illustrates the Treg population within the whole lung after whole lung PAM treatment. *p<0.05, **p<0.005 by ANOVA multiple comparisons. [Figure 16B] Figure 1 illustrates whole lung phenotype by flow cytometry 19 days after ablation of the primary tumor. *p<0.05 by ANOVA multiple comparisons. [Figure 16C] Figure 1 illustrates the characteristics of GzB-secreting T cells. *p<0.05, **p<0.005, ***p<0.0005 by ANOVA multiple comparisons. [Figure 16D] Histological staining of CD8 (brown) and FOXP3 (green) in metastatic lung lesions is illustrated (22x scale). [Figure 16E] Splenic CD45+ cells by flow cytometry 19 days after ablation of the primary tumor are depicted. *p<0.05, ****p<0.0001 in all panels by ANOVA multiple comparisons. [Figure 16F] Splenic CD3+ T cells by flow cytometry are depicted 19 days after ablation of the primary tumor. *p<0.05, ****p<0.0001 in all panels by ANOVA multiple comparisons. [Figure 16G] Figure 1 shows splenic monocytes and monocyte MHC class II expression after treatment. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001 by ANOVA multiple comparisons. [Figure 17] Illustrates the effect of PAM-RT on local and systemic immunomodulation. [Figure 18A] 1 illustrates survival rates of mice treated with no treatment, a single ablation dose or radiation therapy, pre-ablation priming, or post-ablation conditioning, according to some embodiments of the present disclosure. [Figure 18B] 1 illustrates the density of tumor vasculature in mice treated with no treatment, a single ablation dose or radiation therapy, post-ablation modulation, or only four sub-ablation doses, according to some embodiments of the present disclosure. [Figure 19A]1 illustrates tumor growth in C57B16 mice treated with no treatment, a single ablation dose of radiation therapy, or post-ablation modulation, according to some embodiments of the present disclosure. [Figure 19B] 1 illustrates tumor growth in nude mice treated with no treatment, a single ablation dose of radiation therapy, or post-ablation modulation, according to some embodiments of the present disclosure. [Figure 20A] 1 illustrates survival rates of C57B16 mice treated with no treatment, a single ablation dose of radiation therapy, or post-ablation modulation, according to some embodiments of the present disclosure. [Figure 20B] 1 illustrates the survival rate of nude mice treated with no treatment, a single ablation dose of radiation therapy, or post-ablation modulation, according to some embodiments of the present disclosure. [Figure 21A] 1 illustrates leukocyte infiltration of 3LL tumors in C57B16 mice treated with no treatment, a single ablation dose of radiation therapy (24 Gy), post-ablation conditioning, or pre-priming, according to some embodiments of the present disclosure. 1 and 5 days after treatment are also illustrated for mice treated with a single ablation treatment. [Figure 21B] 1 illustrates leukocyte infiltration of 3LL tumors in C57B16 mice treated with no treatment, a single ablation dose of radiation therapy (24 Gy), post-ablation conditioning, or pre-priming, according to some embodiments of the present disclosure. 1 and 5 days after treatment are also illustrated for mice treated with a single ablation treatment. [Figure 21C] 1 illustrates leukocyte infiltration of 4T1 tumors in C57B16 mice treated with no treatment, a single ablation dose of radiation therapy (24 Gy), post-ablation conditioning, or pre-priming, according to some embodiments of the present disclosure. 1 and 5 days after treatment are also illustrated for mice treated with a single ablation treatment. [Figure 22A]1 illustrates the survival rate of mice treated with trabectedin alone, a single ablation dose (24 Gy), post-ablation modulation (22 Gy + 4 × 0.5 Gy), a single ablation dose and trabectedin, or trabectedin and post-ablation modulation, according to some embodiments of the present disclosure. [Figure 22B] 1 illustrates tumor growth in mice treated with trabectedin according to some embodiments of the present disclosure. [Figure 22C] 1 illustrates tumor growth in mice treated with a single ablative dose of radiation therapy and trabectedin, according to some embodiments of the present disclosure. [Figure 22D] 1 illustrates post-ablation modulation and tumor growth in mice treated with trabectedin according to some embodiments of the present disclosure. [Figure 23] Illustrating an experimental program according to some embodiments of the present disclosure, briefly, mice were injected with tumor cells (e.g., 4T1 cells) into the fourth mammary fat pad, and approximately 8 days later, mice received three ablative doses of radiation therapy (3×20 Gy) over three days and anti-PD1 therapeutic agent or vehicle, and 12 days later, some mice were treated with four sub-ablative doses of radiation therapy (4×0.5 Gy) over four days and anti-PD1 therapeutic agent or vehicle. [Figure 24A] 1 illustrates the survival rate of mice injected with D90 cells and treated with three ablation doses of radiation therapy, three ablation doses of radiation therapy and an anti-PD1 therapeutic agent, three ablation doses of radiation therapy and four sub-ablation doses, or three ablation doses of radiation therapy, four sub-ablation doses, and an anti-PD1 therapeutic agent, according to some embodiments of the present disclosure. [Figure 24B] 1 illustrates the survival rate of mice intravenously infused with 4T1 and treated with an anti-PD1 therapeutic, four sub-ablative doses, or four sub-ablative doses and an anti-PD1 therapeutic, according to some embodiments of the present disclosure. [Figure 25]1 illustrates the survival rate of mice injected with 4T1 cells and treated with three ablative doses of radiation therapy, three ablative doses of radiation therapy and an anti-PD1 therapeutic agent, three ablative doses of radiation therapy and four sub-ablative doses, or three ablative doses of radiation therapy, four sub-ablative doses and an anti-PD1 therapeutic agent, according to some embodiments of the present disclosure. [Figure 26] Figure 1 illustrates the significant increase in CD8 / Treg ratios resulting from a significant decrease in Tregs after whole lung PAM treatment. *p<0.05 by ANOVA multiple comparisons. DETAILED DESCRIPTION OF THE INVENTION

[0015] The disclosed methods and devices can treat cancer with an ablative RT dose followed by a sub-ablative RT dose. The ablative RT dose can generate an adaptive immune response, and subsequent sub-ablative RT treatments can affect the tumor microenvironment, causing cancer cells to produce antigenic substances, which can lead to an immunogenic response against the cancer cells. The sub-ablative dose can be used to generate an immune response in delicate tissues while sparing these delicate tissues so that they remain viable after treatment. The disclosed methods and devices are well suited for combination with conventional methods, compounds, and devices for treating cancer. For example, the ablative and sub-ablative doses described herein can be delivered using a radiation therapy system, as known to those skilled in the art. The radiation therapy system can be programmed with software instructions to treat tumors with ablative and sub-ablative doses. The software instructions may include treatment planning software to control the location and timing of the ablative and sub-ablative doses. Alternatively, or in combination, the radiation therapy may include brachytherapy. For example, radioactive seeds may be placed near a tumor, such as a prostate tumor, and sub-ablation treatments can be administered using a radiotherapy device located remotely from the prostate. The methods and devices of the present disclosure can be combined with conventional methods, devices, and compounds for treating cancer, such as, for example, immunomodulatory therapies.

[0016] Without being bound by any particular theory, it is believed that an ablative dose can release a large amount of immunogenic antigens from cancer cells, and subsequent sub-ablative dose treatment can expose cancer cells to sub-ablative doses to present the same antigens, thereby eliciting an immune response. Subsequent sub-ablative dose treatment can also play a role in the tumor microenvironment, reducing the extent to which tumors provide immune privileged sites.

[0017] Different irradiation regimens and doses for tumors can have different immunomodulatory effects. Treatment regimens longer than 7 days are immunosuppressive, while single ablation doses that release large amounts of antigen are immunogenic. The present disclosure provides irradiation schemes for the treatment of solid tumors that increase immunogenicity and tumor accessibility to immune cells and therapeutic agents. In some embodiments, the irradiation schemes described herein combine non-ablation immune priming followed by ablation, referred to as "Immune Priming Ablation" (IPA), which can result in more potent in situ vaccines.

[0018] Conventional therapies are typically ineffective in treating certain solid tumors, in part due to the tumor microenvironment acting as an immune-privileged site, leading to chemotherapy and radiotherapy resistance. Multiple factors contribute to an immunosuppressive tumor microenvironment, including tumor-promoting immune cells, stromal fibrotic responses, and tumor vascular disruption. Tumors can contain abundant immunosuppressive stromal cell populations, including bone marrow-derived suppressor cells (MDSCs), cancer-associated fibroblasts (CAFs), and tumor-associated macrophages (TAMs). TAMs and CAFs may play a key role in shaping excess extracellular matrix (ECM) by collaborating to induce a fibrogenic response, similar to a "wound healing" response after injury. Tumor-associated macrophages constitute the majority of resident immune cells in pancreatic cancer, affecting the inhibition of infiltrating cytotoxic T cells and can typically be characterized as anti-tumorigenic M1 and pro-tumorigenic M2. Dendritic cells (DCs), a very small immune cell population, are resident in tumors and are often tolerogenic, and they also act as immunosuppressive regulatory T cells (T regThis leads to the induction of inflammatory cytokines (IGCs) and the inhibition of cytotoxic T cells. Many tumor cells downregulate the expression of major histocompatibility complex (MHC) class I and components of the antigen presentation machinery, essentially preventing MHC peptide presentation and thereby evading recognition by immune effector cells. The interstitial fibrotic response leads to the formation of trabecular meshwork, which further inhibits the access of cytotoxic immune cells and separates tumor cells from blood vessels, while also reducing permeability.

[0019] Disrupted and inefficient tumor vasculature may also play a role in perpetrating the immunosuppressive tumor microenvironment, which is partially due to uncontrolled tumor growth. As tumors progress, angiogenesis often fails to keep pace with growth rates, and thus a structural vascular network cannot be efficiently formed. Tumor vasculature is also characterized by immature and leaky blood vessels, contributing to the increased interstitial pressure seen in many solid tumors, which can lead to reduced immune cell extravasation. This disruption can help lead to chemotherapy and radiotherapy resistance through increased hypoxia and lack of proximity to drugs as well as immune infiltrates.

[0020] Radiation therapy has a well-established role in local tumor control through direct cell death. Anecdotal evidence suggests that tumor ablation may occasionally produce reactions at distant sites, which are hypothesized to arise from eliciting systemic immunity. Radiation has been shown to induce immunogenic cell death (ICD), which may be the first step in antitumor immunity. Damage and cellular stress caused by radiation therapy can promote the activation and maturation of dendritic cells (DCs). DCs are antigen-presenting cells (APCs) that take up antigens from their environment and present them to T cells at MHC class I or II receptors. T cell activation, determined not only by antigen presentation from DCs but also by costimulatory molecules, is one of the most important steps in adaptive-dependent antitumor immunity. Due to the immunosuppressive tumor microenvironment, T cell activation is often ineffective. Harnessing radiation can lead to much more effective clinical outcomes by activating potential while suppressing adverse effects according to the embodiments described herein.

[0021] There are three clinically applicable dosing regimens for cancer treatment: conventional fractionation, sub-ablative hypofractionation, and ablative hypofractionation (Table 1). These conventional dosing regimens can be combined with sub-ablative therapy in some embodiments. Conventional fractionation in cancer treatment consists of multiple low-dose fractions delivered over a longer period (greater than 7 days) and is generally immunosuppressive, thought to repeatedly kill radiosensitive infiltrating immune cells. Sub-ablative hypofractionation consists of larger non-lethal doses delivered over less than 7 days and has some immunomodulatory effects. This regimen enhances anti-tumor immune responses, but the cytotoxicity of the treatment alone is not very effective in controlling tumor growth. Ablative hypofractionation can cause direct cell death, release large amounts of antigens, and control local tumor growth. The drawback of a single ablative dose of RT is that it induces a pro-tumor fibrogenic response orchestrated, in part, by tumor-associated macrophages of a tumorigenic phenotype that secrete TGFβ, and attracts immunosuppressive cells into the tumor microenvironment, which may reduce the anti-tumor response. The effects of various forms of sub-radiotherapy are summarized in Tables 1 and 2, which are suitable for combination according to some embodiments.

[0022] Treating tumors using a combination of ablation therapy (e.g., a single ablation therapy) and one or more sub-ablative therapy sessions can combine the benefits of both ablation therapy, which generates antigens and kills cells, and sub-ablative therapy, which increases immune proximity to the tumor site and activates the immune system. The initial ablation therapy results in enhanced release of tumor antigens, increased expression of damage-associated molecular patterns, and upregulation of MHC class I on surviving tumor cells, thereby reversing tumor immune escape. Subsequent use of one or more sub-ablative doses over a short period, e.g., less than seven days, can provide additional immunomodulatory benefits, including normalization of vasculature, upregulation of damage-associated molecular patterns, MHC class I, and adhesion markers, and enhanced release of chemokines, thereby 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, resulting in a therapeutic effect far greater than would be expected from either therapy alone. The hypothesized effects of this treatment are summarized in Table 2.

[0023] [Table 1]

[0024] [Table 2]

[0025] FIG. 1 illustrates a radiation therapy treatment system (10) capable of delivering radiation therapy to a patient (14) as described herein. The radiation therapy system (10) may include one or more components of numerous conventional systems suitable for combination in accordance with the embodiments disclosed herein. Examples of conventional systems suitable for combination in accordance with the present disclosure include Accuray systems such as CyberKnife, Radixact, and TomoTherapy treatment systems, as well as Varian systems 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 for aligning the patient and tumor. The radiation therapy treatment may include photon-based radiation therapy, brachytherapy, electron beam therapy, proton, neutron, or particle beam therapy, or other types of therapeutic modalities. The treatment system (10) includes a digital processing unit (601) for controlling the energy level and dose of beams delivered to a subject, for example, using stereotactic radiotherapy (STRT). The treatment system (10) may also include imaging components and systems as known to those skilled in the art. The radiation therapy treatment system (10) includes a gantry (18) coupled to a computer to control beam placement, although other beam directing devices may be used. The gantry (18) may 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 beam of radiation (30). The gantry (18) shown in the figures is a circular gantry, i.e., extends through a full 360° arc to form a complete circle or ring, although other types of mounting arrangements may be employed. For example, a C-shaped, partial circular gantry, or a robotic arm may be used.Any other framework capable of positioning the radiation modules 22 at various rotational and / or axial positions relative to the patient 14 may be employed. Additionally, the radiation source 24 may travel along a path that does not follow the shape of the gantry 18. For example, while the illustrated gantry 18 is generally annular, the radiation source 24 may travel along a path that is not annular.

[0026] The radiation module 22 may also include a conditioning device 34 operable to modify or adjust the radiation beam 30. The conditioning device 34 provides conditioning of the radiation beam 30 and directs the radiation beam 30 toward the patient 14. Specifically, the radiation beam 34 is directed toward a portion of the patient. Generally, this 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 desired to receive radiation is an example of a region of interest. The region of interest 38 may include a first treatment site, a second treatment site, or a subsequent treatment site. The region of interest 38 may also be peripheral or partially peripheral to the subject. Another type of region of interest is a region at risk of radiation damage. If the portion includes a region at risk of radiation damage, the radiation beam is preferably dispersed from the region. The patient 14 may have more than one region receiving radiation therapy as described herein.

[0027] FIG. 2A illustrates an example of a patient having a tumor in a first region, in this case the liver. This tumor may be treated using the methods described herein by administering an ablation dose of radiation therapy to all or a portion of the tumor, followed by administering one or more sub-ablative doses of radiation therapy to the same tissue. In some cases, the disclosed methods may contemplate administering a first ablation dose of radiation therapy to all or a portion of the tumor, followed by administering one or more sub-ablative doses to the tumor or to another location(s) in the subject. The sub-ablative doses may be administered to all or a portion of the tumor. If an ablation dose is administered to a portion of the tumor, the sub-ablative doses may be administered to portions of the same tumor or to different portions of the tumor.

[0028] Sub-ablation doses administered following an ablation dose of radiation therapy can enhance the immune response to the tumor and enhance the therapeutic response. For example, as shown in FIG. 7A, tumor-bearing mice received either no treatment, a single ablation dose of radiation therapy to the tumor, or a single ablation dose of radiation therapy to the tumor followed by up to four sub-ablation doses of radiation therapy. Mice receiving an ablation dose followed by a sub-ablation dose had improved survival rates. Mice receiving the ablation dose and sub-ablation dose in the reverse order, i.e., four sub-ablation doses followed by a single ablation dose, had slightly better survival rates than mice treated with only a single ablation dose, but not as good as when the ablation dose was administered before the sub-ablation dose. Similar approaches can be used for the treatment of patients, such as humans, and animal patients, for example, using appropriate software instructions for a radiation therapy system such as those described herein.

[0029] FIG. 2B illustrates an example of a patient with two tumors in an organ, such as the lung. The two tumors may include a first tumor and a second tumor. The first tumor may include a primary tumor, and the second tumor may include a metastatic or recurrent tumor. Alternatively, the first tumor may include a metastatic or recurrent tumor, and the second tumor may include a primary tumor. Using the methods described herein, the first tumor can be treated by administering an ablative dose of radiation therapy to all or a portion of the tumor. The first and second tumors may then each be treated with one or more sub-ablative doses of radiation therapy. Subsequent sub-ablative doses of radiation therapy 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 shorten its length despite not receiving an ablative dose. Using the methods described herein, a patient may have a first tumor and any number of second tumors. A first ablative dose of radiation therapy may be administered to a first tumor, followed by one or more sub-ablative doses of radiation therapy administered to some or all of a second tumor. The sub-ablative doses of radiation therapy may also be administered to the first tumor after the administration of the ablative dose of radiation therapy. The second tumor may be in the same organ as the tumor being treated with the ablative dose, or may be in one or more different organs. In some cases, the first tumor is selected based on its size, location, disease stage, or other clinically relevant characteristics.

[0030] FIG. 3 illustrates an example of a patient with a tumor in the lung and a metastatic tumor in the bone. In some cases, the tumor in the lung may be treated with a single ablation dose of radiation therapy. The entire lung may be treated with one or more sub-ablative doses of radiation therapy after the first dose. While the entire lung is being treated with one or more sub-ablative doses, the bone containing the metastatic tumor may be treated with one or more sub-ablative doses. In another example, the location of the metastatic bone tumor may be treated first with a single ablation dose of radiation therapy. After this first ablation dose, the metastatic bone tumor and either the tumor in the lung or the entire lung may be treated with one or more sub-ablative doses.

[0031] In some cases, after treatment of a first tumor with an ablation dose, one or more sub-ablative doses may be administered systemically. In some cases, one or more sub-ablative doses may be administered, for example, to the entire abdominal cavity. In some cases, one or more sub-ablative doses may be administered to an organ known to contain one or more metastatic tumors or to an organ known to be prone to developing 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 another lung, or in the adrenal gland, bone, brain, and liver. Examples of organs prone to developing metastatic tumors include, but are not limited to, bone, lymph nodes, lung, liver, brain, adrenal gland, breast, eye, kidney, muscle, pancreas, salivary gland, and spleen.

[0032] FIG. 4 illustrates an exemplary process (400) that can be used to treat cancer. The first step (405) of the process involves identifying a first treatment region. The first treatment region may include a region of a tumor within a 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 a patient, the first treatment region may include the location of a tumor where radiation therapy is most available. In some cases, if several different tumors are present in a patient, the first treatment region may be the location of a tumor that is safest to treat with radiation. A tumor that is safest to treat with radiation may be located in a position that is more likely to reduce 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.

[0033] After the first region is identified, a second treatment region is identified in step (410). In some embodiments, the second treatment region may include a location having 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 than the first treatment region. The second treatment region may be a location having a secondary or primary tumor. In some cases, the second treatment region has a smaller tumor than the first treatment region. In some cases, the second treatment region is a location that is difficult to treat with radiation. In some cases, the second treatment region is a location that is more sensitive to the adverse effects of radiation therapy than the first treatment region. In some cases, the second treatment region is a location that does not have an identified tumor but is known to be prone to developing metastatic tumors.

[0034] The next step (415) involves registering the radiation therapy system and the target patient at the first treatment region. The radiation therapy system may include any radiation therapy system components known in the art, including known radiation therapy systems commercially available from Varian Medical and Accuray. For example, the radiation therapy system may include the system of FIG. 1 or a similar system. The type of radiation therapy may include any type suitable for clinical use as described herein, including, for example, x-ray radiation, gamma radiation, alpha particle radiation, beta particle radiation, neutron particle radiation, external beam radiation, or brachytherapy. For example, the radiation therapy system may include an imaging system capable of imaging and targeting a tumor within a patient.

[0035] Once the first treatment region is registered, it can be treated with an ablative dose of radiation in step (420). The ablative dose may be between about 10 Gy and about 60 Gy, between about 20 Gy and about 40 Gy, or between about 20 Gy and about 30 Gy. In some cases, the ablative 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 a range defined by any two of the foregoing values.

[0036] Following the first treatment, step (425) of process (400) requires waiting a predetermined period of time before proceeding with a subsequent treatment. For example, the predetermined period of time may be between about 1 hour and about 4 days. In some cases, the predetermined period of time may be between about 1 hour and about 36 hours, between about 6 hours and about 30 hours, between about 12 hours and about 26 hours, between about 20 hours and about 28 hours, or between about 22 hours and about 26 hours. One of ordinary skill in the art may determine the appropriate waiting period following the teachings of the present disclosure.

[0037] In step (430), the radiation therapy system is registered at a second treatment region, and in step (435), the second treatment region can be treated with a sub-ablative dose. The sub-ablative dose may include a dose between about 0.1 Gy and about 3 Gy, between about 0.2 Gy and about 2 Gy, between about 0.3 Gy and about 1 Gy, between about 0.3 Gy and about 0.7 Gy, between about 0.1 Gy and about 0.5 Gy, or between about 0.8 Gy and about 1.2 Gy. Steps (425) through (435) may be repeated multiple times. In some cases, steps (425) through (435) are repeated two, three, four, five, six, or more than six times to elicit an immunogenic response in the second treatment region or additional sub-ablative treatment regions without ablation. In some cases, the sub-ablation dose includes multiple sub-ablation doses, each of which includes a dose between about 0.1 Gy and about 3 Gy, between about 0.2 Gy and about 2 Gy, between about 0.3 Gy and about 1 Gy, between about 0.3 Gy and about 0.7 Gy, between about 0.1 Gy and about 0.5 Gy, or between about 0.8 Gy and about 1.2 Gy. In step (440), the radiation therapy system may then be registered with a third treatment region, and in step (445), the third treatment region is treated with the sub-ablation dose. The sub-ablation dose may be a dose between about 0.1 Gy and about 3 Gy, between about 0.2 Gy and about 2 Gy, between about 0.3 Gy and about 1 Gy, between about 0.3 Gy and about 0.7 Gy, or between about 0.8 Gy and about 1.2 Gy. Steps (440) and (445) may be repeated any number of times if beneficial to elicit an immunogenic response without ablation in a third treatment area.

[0038] After performing treatment in the third treatment area, the system may wait (450) a predetermined period of time, and then repeat steps (440)-(450) one, two, three, four, or more than four times. The predetermined period of time may be between about 1 hour and about 4 days. In some cases, the predetermined period of time may be between about 1 hour and about 36 hours, between about 6 hours and about 30 hours, between about 12 hours and about 26 hours, between about 20 hours and about 28 hours, or between about 22 hours and about 26 hours.

[0039] A processor such as those described herein may be configured with instructions to perform the method (400).

[0040] While Figure 4 illustrates a method (400) for treating cancer according to one embodiment, those skilled in the art will recognize that numerous adaptations and variations are possible. One or more of the steps may be omitted, repeated, performed simultaneously, and / or performed in a different order. In some embodiments, one or more of the steps may be modified or include substeps. Additionally, those skilled in the art will understand that additional steps may be included when practicing this method.

[0041] In some cases, the cumulative amount of radiation therapy delivered to the second region over the course of treatment comprises a sub-ablative dose. In some cases, the cumulative sub-ablative dose comprises multiple sub-ablative doses.

[0042] FIG. 5A illustrates in vivo treatment, which, according to some embodiments, may be achieved by pre-priming a tumor with one or more sub-ablative doses of radiotherapy prior to administering an ablative dose of radiotherapy. In this case, a tumor, depicted with blood vessels and immune cells (black circle), is first treated with four sub-ablative doses (e.g., 0.5 Gy or 1 Gy, depicted by small "lightning bolts"), followed by a single ablative dose (e.g., 22 Gy or 20 Gy, depicted by a large "lightning bolt"). The pre-priming sub-ablative dose may sensitize the tumor to immune cells. This may be evidenced by increased expression of stress markers, immunomodulatory / immunogenic cell death, and differential expression of peptide reservoirs and MHC class I. The tumor is then treated with an ablative dose of radiotherapy, which results in cell death, particularly of tumor cells, releasing tumor antigens into the tumor microenvironment and circulation. Ablative radiation therapy also induces other changes in tumor viability cells, such as increased expression of damage-associated molecular patterns and increased release of cytokines, which promote immunogenicity. The release of tumor antigens stimulates effector T-cell responses, which are driven by tumor sensitization achieved by the prior subablative dose.

[0043] Figure 5B illustrates an example of post-ablation modulation and associated bioprocessing. In this case, a tumor, indicated by blood vessels and immune cells (black circle), is first treated with a single ablation dose (e.g., 22 Gy or 20 G, depicted by the large "lightning bolt"), followed by four sub-ablation doses (e.g., 0.5 Gy or 1 Gy, depicted by the small "lightning bolt"). The first ablation dose of radiation therapy induces cell death and release of antigens that stimulate effector T cell responses. Subsequent sub-ablation doses of radiation therapy modulate the tumor environment, increasing the infiltration of activated effector T cells into the tumor. This increased infiltration is driven by cellular changes induced by the sub-ablation treatment, including normalization of the vasculature (see Figure 7B), changes in the cytokine environment, and macrophage repolarization / reprogramming. As shown in Figure 7A, both the pre-priming treatment and post-ablation modulation increased the survival rate of tumor-bearing mice. However, post-ablation modulation showed a much greater impact on survival. Figure 17 provides a summary of the effect of PAM-RT treatment on local and systemic immunomodulation.

[0044] The methods and systems disclosed herein may be used to treat patients. In some embodiments, the patient is human. In some embodiments, the patient has been diagnosed with cancer. In some embodiments, the patient has been diagnosed with a solid tumor. Examples of cancer types include, but are not limited to, esophageal cancer, breast cancer, gastric cancer, intrahepatic 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 a primary tumor in an organ. Examples of organs that may develop cancer include, but are not limited to, 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 may have at least one detected tumor. In some cases, the patient's cancer has not responded to or has ceased to respond to another treatment.

[0045] In some embodiments, described herein are methods of treating cancer in a subject, the methods comprising delivering an ablation dose of radiation therapy to a first region containing the cancer, followed by a sub-ablative dose to a second region, wherein the sub-ablative dose is administered after the ablation dose. In some embodiments, the sub-ablative dose is administered at least one hour after the ablation dose. In some embodiments, the sub-ablative dose is administered at least one day after the ablation dose. In some embodiments, the sub-ablative dose is administered at most four days after the ablation dose.

[0046] Digital Processing Unit In some examples, the platforms, systems, media, and methods described herein include, or use of, a digital processing device. 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 still further embodiments, the digital processing device further includes an operating system configured to execute the executable instructions. In some embodiments, the digital processing device is optionally connected to a computer network. In further embodiments, the digital processing device is optionally connected to the Internet to access the World Wide Web. In still further embodiments, the digital processing device is optionally connected to a cloud computing infrastructure. 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.

[0047] In accordance with the description herein, suitable digital processing devices include, by way of non-limiting example, server computers, desktop computers, laptop computers, notebook computers, subnotebook computers, netbook computers, netpad computers, set-top computers, media streaming terminals, handheld computers, Internet appliances, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles. Those skilled in the art will recognize that many smartphones are suitable for use with the systems described herein. Those skilled in the art will recognize that selected televisions, video players, and digital music players, optionally with computer network connectivity, are suitable for use with the systems described herein. Suitable tablet computers include those with booklet, slate, and convertible configurations known to those skilled in the art.

[0048] In some embodiments, the digital processing device includes an operating system configured to execute executable instructions. An operating system is software, including programs and data, that controls the device's hardware and provides services for the execution of applications, for example. Those skilled in the art will recognize that suitable server operating systems include, by way of non-limiting example, FreeBSD, OpenBSD, NetBSDR®, Linux, Apple® Mac OS X Server®, Oracle® Solaris®, Windows Server®, and Novell® NetWare®. Those skilled in the art will recognize that suitable personal computer operating systems include, by way of non-limiting example, Microsoft® Windows®, Apple® Mac OS X®, UNIX®, and UNIX-like operating systems, such as GNU / Linux®. In some embodiments, the operating system is provided by cloud computing. Those skilled in the art will also recognize that suitable mobile smartphone operating systems include, by way of non-limiting example, Nokia® Symbian® OS, Apple® iOS®, Research In Motion® BlackBerry® OS, Google® Android®, Microsoft® Windows Phone® OS, Microsoft® Windows Mobile® OS, Linux®, and Palm® WebOS®.Those skilled in the art will also recognize that suitable media streaming device operating systems include, by way of non-limiting example, Apple TV®, Roku®, Boxee®, Google TV®, Google Chromecast®, Amazon Fire®, and Samsung® HomeSync®. Those skilled in the art will also recognize that suitable video game console operating systems include, by way of non-limiting example, Sony® PS3®, Sony® PS4®, Microsoft® Xbox 360®, Microsoft Xbox One, Nintendo® Wii®, Nintendo® Wii U®, and Ouya®.

[0049] In some embodiments, the device includes a storage device and / or memory device. A storage device and / or memory device is one or more physical devices used to temporarily or permanently store data or programs. In some embodiments, the device is volatile memory and requires power to maintain stored information. In some embodiments, the device is nonvolatile memory and retains stored information even when power is not supplied to the digital processing device. In further embodiments, the nonvolatile memory includes flash memory. In some embodiments, the nonvolatile memory includes dynamic random access memory (DRAM). In some embodiments, the nonvolatile memory includes ferroelectric random access memory (FRAM). In some embodiments, the nonvolatile memory includes phase change random access memory (PRAM). In other embodiments, the device is a storage device, including, by way of non-limiting examples, CD-ROMs, DVDs, flash memory devices, magnetic disk drives, magnetic tape drives, optical disk drives, and cloud computing-based storage devices. In further embodiments, the storage device and / or memory device is a combination of devices such as those disclosed herein.

[0050] In some embodiments, the digital processing device includes a display for conveying 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 further embodiments, 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 still further embodiments, the display is a combination of devices such as those disclosed herein.

[0051] 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 way of non-limiting example, a mouse, trackball, trackpad, joystick, game controller, or stylus. In some embodiments, the input device is a touchscreen or multi-touchscreen. In other embodiments, the input device is a microphone for capturing voice or other audio input. In other embodiments, the input device is a video camera or other sensor for capturing motion or visual input. In further embodiments, the input device is a Kinect, Leap Motion, or the like. In still further embodiments, the input device is a combination of devices, such as those disclosed herein.

[0052] Referring to FIG. 6 , in certain embodiments, an exemplary digital processing device (601) is programmed or otherwise configured into a radiation therapy device as described herein. The device (601) can control various aspects of the radiation therapy device of the present disclosure, such as performing processing steps. In this embodiment, the digital processing device (601) includes a central processing unit (CPU, also referred to herein as a “processor” and “computer processor”) (605), which may be a single-core or multi-core processor, or multiple processors for parallel processing. The digital processing device (601) also includes memory or storage locations (610) (e.g., random access memory, read-only memory, flash memory), electronic storage (615) (e.g., a hard disk), communication interfaces (620) (e.g., a network adapter) for communicating with one or more other systems, and peripherals (625), such as cache, other memory, data storage, and / or electronic display adapters. The memory (610), storage (615), interface (620), and peripherals (625) communicate with the CPU (605) via a communication bus (solid lines), such as a motherboard. The storage (615) may be a data storage device (or data repository) for storing data. The digital processing device (601) may be operatively connected to a computer network ("network") (630) utilizing the communication interface (620). The network (630) may be the Internet and / or an extranet, or an intranet and / or extranet in communication with the Internet. In some cases, the network (630) is a telecommunications and / or data network. The network (630) may include one or more computer servers, which may enable distributed computing, such as cloud computing.The network (630) may, in some cases, utilize the device (601) to implement a peer-to-peer network, which may allow devices coupled to the device (601) to act as clients or servers.

[0053] Continuing with reference to FIG. 6, the CPU (605) may execute a sequence of machine-readable instructions embodied in a program or software. The instructions may be stored in a memory location, such as memory (610). The instructions may be directed to the CPU (605), which may then program or otherwise configure the CPU (605) to perform the methods of the present disclosure. Examples of operations performed by the CPU (605) may include fetch, decode, execute, and writeback. The CPU (605) may be part of a circuit, such as an integrated circuit. One or more other components of the device (601) may also be included in the circuit. In some embodiments, the circuit is an application-specific integrated circuit (ASIC) or a field-programmable gate array (FPGA).

[0054] 6, the storage device (615) can store files such as drivers, libraries, and saved programs. The storage device (615) can store user data such as user preferences and user programs. In some cases, the digital processing device (601) can include one or more additional data storage devices external to the device, such as those located on remote servers in communication over an intranet or the Internet.

[0055] Continuing with reference to Figure 6, the digital processing device (601) can communicate with one or more remote computer systems over a network (630). For example, the device (601) can communicate with a user's remote computer system. Examples of remote computer systems include a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., an Apple® iPad, a Samsung® Galaxy Tab), a telephone, a smartphone (e.g., an Apple® iPhone, an Android-enabled device, a Blackberry®), or a personal digital assistant.

[0056] Methods as described herein can be performed by machine (e.g., a computer processor) executable code stored in electronic storage locations of the digital processing device (601), such as, for example, memory (610) or electronic storage (615). The machine-executable or machine-readable code can be provided in the form of software. In use, the code is executable by the processor (605). In some cases, the code can be retrieved from storage (615) and stored in memory (610) for easy access by the processor (605). In some situations, the electronic storage (615) can be omitted, and the machine-executable instructions are stored in memory (610).

[0057] In some embodiments, disclosed herein is a computer-readable medium configured with instructions that, when executed, cause a processor to provide instructions to a radiation therapy system to deliver an ablation dose of radiation therapy to a first region and deliver a sub-ablative dose to a second region following the ablation dose. In some embodiments, the sub-ablative dose is delivered at least one hour after the ablation dose. In some embodiments, the sub-ablative dose is delivered at least one day after the ablation dose. In some embodiments, the sub-ablative dose is delivered at most four days after the ablation dose.

[0058] Non-transitory computer-readable storage medium In some examples, the platforms, systems, media, and methods disclosed herein include one or more non-transitory computer-readable storage media encoded with a program including 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 is optionally removable from the digital processing device. In some embodiments, computer-readable storage media include, by way of non-limiting example, CD-ROMs, DVDs, flash memory devices, solid-state storage devices, magnetic disk drives, magnetic tape drives, optical disk drives, cloud computing systems and services, and the like. In some cases, the programs and instructions are encoded in the medium permanently, substantially permanently, semi-permanently, or non-transitoryly.

[0059] computer program In some embodiments, the platforms, systems, media, and methods disclosed herein include at least one computer program, or the use thereof. A computer program includes a sequence of instructions written to perform specific tasks and executable by a CPU of a digital processing device. The computer-readable instructions may be implemented as program modules, such as functions, objects, application programming interfaces (APIs), data structures, etc., that perform specific tasks or implement specific abstract data types. In light of the disclosure provided herein, those skilled in the art will recognize that computer programs may be written in a variety of languages ​​and in a variety of styles.

[0060] The functionality of the computer-readable instructions may be combined or distributed as desired in various environments. In some embodiments, a computer program includes one sequence of instructions. In some embodiments, a computer program includes multiple sequences of instructions. In some embodiments, a computer program is provided in one location. In other embodiments, a computer program is provided in multiple locations. In various embodiments, a computer program includes one or more software modules. In various embodiments, a computer program, in part or in whole, includes one or more web applications, one or more mobile applications, one or more standalone applications, one or more web browser plug-ins, extensions, add-ins, or add-ons, or any combination thereof.

[0061] Web Applications In some embodiments, the computer program comprises a web application. In light of the disclosure provided herein, those skilled in the art will recognize that in various embodiments, a web application utilizes one or more software frameworks and one or more database systems. In some embodiments, the web application is created in a software framework such as Microsoft® .NET or Ruby on Rails (RoR). In some embodiments, the web application utilizes one or more database systems, including, by way of non-limiting example, relational, non-relational, object-oriented, associative, and XML database systems. In further embodiments, suitable relational database systems include, by way of non-limiting example, Microsoft® SQL Server, mySQL™, and Oracle®. Those skilled in the art will also recognize that in various embodiments, a web application is written in one or more languages ​​and in one or more styles. A web application may 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 client-side scripting language such as Asynchronous Javascript and XML (AJAX), Flash® Actionscript, Javascript, or Silverlight®.In some embodiments, the web application is written to some extent in a server-side coding language such as Active Server Pages (ASP), ColdFusion®, Perl, Java™, JavaServer Pages (JSP), Hypertext Preprocessor (PHP), Python™, Ruby, Tcl, Smalltalk, WebDNA®, or Groovy. In some embodiments, the web application is written to some extent in a database query language such as Structured Query Language (SQL). In some embodiments, the web application integrates with enterprise server products such as IBM® Lotus Domino®. In some embodiments, the web application includes a media player element. In various further embodiments, the media player element utilizes one or more of a number of suitable multimedia technologies, including, by way of non-limiting example, Adobe® Flash®, HTML 5, Apple® QuickTime®, Microsoft® Silverlight®, Java™, and Unity®.

[0062] Mobile Applications In some embodiments, the computer program comprises a mobile application provided to the mobile digital processing device. In some embodiments, the mobile application is provided to the mobile digital processing device at the time of manufacture. In other embodiments, the mobile application is provided to the mobile digital processing device over a computer network as described herein.

[0063] In light of the disclosure provided herein, mobile applications are created using hardware, languages, and development environments known in the art and techniques known to those skilled in the art. Those skilled in the art will recognize that mobile applications may be written in multiple languages. Suitable programming languages ​​include, by way of non-limiting example, C, C++, C#, Objective-C, Java™, Javascript, Pascal, Object Pascal, Python™, Ruby, VB.NET, WML, and XHTML / HTML with or without CSS, or combinations thereof.

[0064] Suitable mobile application development environments are available from several sources. Commercially available development environments include, by way of non-limiting example, Airplay SDK, alcheMo, Appcelerator®, Celsius, Bedrock, Flash Lite, .NET Compact Framework, Rhomobile, and WorkLight Mobile Platform. Other development environments are available free of charge and include, by way of non-limiting example, Lazarus, MobiFlex, MoSync, and Phonegap. Mobile device manufacturers also distribute software development kits, including, by way of non-limiting example, iPhone and iPad (iOS) SDK, Android™ SDK, BlackBerry® SDK, BREW SDK, Palm® OS SDK, Symbian SDK, webOS SDK, and Windows® Mobile SDK.

[0065] Those skilled in the art will recognize that several commercial forums are available for the distribution of mobile applications, including, by way of non-limiting example, the Apple® App Store, Google® Play, Chrome WebStore, BlackBerry® App World, the App Store for Palm devices, the App Catalog for webOS, the Windows® Marketplace for Mobile, the Ovi Store for Nokia® devices, Samsung® Apps, and the Nintendo® DSi Shop.

[0066] Standalone Applications In some embodiments, a computer program includes a standalone application, which is a program that runs as an independent computer process and is not an add-on, e.g., a plug-in, to an existing process. Those skilled in the art will recognize that standalone applications are often compiled. A compiler is a computer program that converts source code written in a programming language into binary object code, such as assembly language or machine code. Suitable compiled programming languages ​​include, by way of non-limiting example, C, C++, Objective-C, COBOL, Delphi, Eiffel, Java™, Lisp, Python™, Visual Basic, and VB.NET, or combinations thereof. Compilation is often performed, at least in part, to create an executable program. In some embodiments, a computer program includes one or more executable compiled applications.

[0067] Web browser plugin In some embodiments, the computer program comprises a web browser plug-in (e.g., an extension). In computing, a plug-in is one or more software components that add specific functionality to a larger software application. Software application manufacturers support plug-ins to enable third-party developers to create capabilities that extend the application, support the easy addition of new functionality, and reduce the size of the application. When supported, plug-ins allow for customization of the functionality of a software application. For example, plug-ins are commonly used in web browsers to play video, create interactivity, scan for viruses, and display specific file types. Those skilled in the art will be familiar with several web browser plug-ins, including Adobe® Flash® Player, Microsoft® Silverlight®, and Apple® QuickTime®. In some embodiments, the toolbar comprises one or more web browser extensions, add-ins, or add-ons. In some embodiments, the toolbar comprises one or more explorer bars, tool bands, or desk bands.

[0068] In light of the disclosure provided herein, those skilled in the art will recognize that several plug-in frameworks are available that allow for plug-ins to be developed in a variety of programming languages, including, by way of non-limiting example, C++, Delphi, Java™, PHP, Python™, and VB .NET.

[0069] A web browser (also called an Internet browser) is a software application designed for use with networked digital processing devices to search, display, and traverse information resources on the World Wide Web. Suitable web browsers include, by way of non-limiting example, Microsoft® Internet Explorer®, Mozilla® Firefox®, Google® Chrome, Apple® Safari®, Opera Software® Opera®, and KDE Konqueror. In some embodiments, the web browser is a mobile web browser. Mobile web browsers (also called microbrowsers, minibrowsers, and wireless browsers) are designed for use on mobile digital processing devices, including, by way of non-limiting example, handheld computers, tablet computers, netbook computers, subnotebook computers, smartphones, music players, personal digital assistants (PDAs), and handheld video game systems. Suitable mobile web browsers include, by way of non-limiting example, Google® Android® Browser, RIM BlackBerry® Browser, Apple® Safari®, Palm® Blazer, Palm® WebOS® Browser, Mozilla® Firefox® for mobile, Microsoft® Internet Explorer® Mobile, Amazon® Kindle® Basic Web, Nokia® Browser, Opera Software® Opera® Mobile, and Sony® PSP™ Browser.

[0070] Software Module In some embodiments, the platforms, systems, media, and methods disclosed herein include software, server, and / or database modules, or the use thereof. In light of the disclosure provided herein, software modules are created by techniques known to those skilled in the art using machines, software, and languages ​​known in the art. The software modules disclosed herein are implemented in many ways. In various embodiments, a software module comprises a file, a portion of code, a programming object, a programming structure, or a combination thereof. In further various embodiments, a software module comprises multiple files, multiple portions of code, multiple programming objects, multiple programming structures, or a combination thereof. In various embodiments, one or more software modules include, by way of non-limiting examples, a web application, a mobile application, and a standalone application. In some embodiments, a software module is in one computer program or application. In other embodiments, a software module is in more than one computer program or application. In some embodiments, a software module is hosted on one machine. In other embodiments, a software module is hosted on more than one machine. In further embodiments, a software module is hosted on a cloud computing platform. In some embodiments, the software modules are hosted on one or more machines in one location. In other embodiments, the software modules are hosted on one or more machines in more than one location.

[0071] Database In some embodiments, the platforms, systems, media, and methods disclosed herein include, or the use of, one or more databases. In light of the disclosure provided herein, those skilled in the art will recognize that numerous databases are suitable for storing and retrieving information. In various embodiments, suitable databases include, by way of non-limiting example, relational databases, non-relational databases, object-oriented databases, object databases, entity-relationship model databases, associative 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 web-based. In still further embodiments, the database is cloud computing-based. In other embodiments, the database is one or more local computer storage devices.

[0072] Example 1 Localized PAM-RT Delays Tumor Growth and Increases Survival in Mice To investigate the efficacy of PAM-RT in local tumor control, C57BL / 6 mice bearing palpable subcutaneous 3LL tumors were divided into five treatment cohorts for a pilot study: untreated; 24 Gy on days 1 or 5 of treatment; 20 Gy after 1 Gy x 4; or 20 Gy after 1 Gy x 4. Essentially, four 1 Gy fractions of RT administered to the primary tumor either before or after a single 20 Gy fraction were compared with a single 24 Gy fraction administered with RT. As shown in Figure 7A-D, compared with a single dose of RT, pretreatment with a low dose of RT demonstrated minimal tumor control, whereas PAM-RT with 1 Gy x 4 fractions significantly delayed tumor growth and improved survival.

[0073] Because 3LL tumors grow rapidly, differences in tumor size may have affected the efficacy of priming RT during ablative RT on day 5 of treatment. Further studies of tumor growth led us to adopt PAM-RT using 0.5 Gy × 4 fractions as the optimal adjusted dose in all further experiments. As before, we compared a single dose of 24 Gy ablative fractionation with PAM-RT using 22 Gy followed by four 0.5 Gy fractions. The dosing regimen is outlined in Figure 8A. Compared to 24 Gy, PAM-RT delayed tumor growth, as seen in the growth curves in Figure 8B and the volume tripling times shown in Figure 8C. Furthermore, PAM-RT significantly improved the survival rate of these animals, as seen in Figure 8D. Although a single fraction of 24 Gy cannot cure 3LL tumor growth, significant growth delay was observed after PAM-RT in mice bearing large 3LL tumors, where single-fraction ablative RT had little effect. Finally, the efficacy of PAM-RT was lost in immunodeficient (nude) mice. Figure 8E shows tumor growth in 3LL tumor-bearing nude mice, and Figure 8F shows the survival rate of these mice. These data demonstrate that PAM can delay local tumor progression and significantly increase survival rates in 3LL tumor-bearing mice, although all mice eventually succumbed to local tumor growth. Furthermore, the efficacy of PAM-RT was contingent on a healthy immune system.

[0074] Immunomodulatory effects of topical PAM-RT in vitro We also investigated the immunomodulatory effects of PAM-RT on 3LL cells in vitro. The experimental scheme is shown in Figure 9A. Six hours after treatment, no difference in cell death was observed; however, after 24 hours, PAM-RT induced significantly more cell death than ablation alone (Figure 9B). Compared with untreated cells or cells treated with low doses of radiation, ablative radiation with or without PAM-RT increased cell surface immunomodulatory markers (CD80), stress markers (CRT, Hsp70, Fas, and MHCI), and immunosuppressive markers (CD47 and PDL1) at 6 hours after RT. Interestingly, as seen in Figure 9C, after 24 hours, cells treated with PAM-RT showed stronger cell surface expression of 4-1BBL, CRT, Fas, Hsp70, and PD-L1 compared to those treated with ablation alone.

[0075] To examine the effects of PAM-RT on immune cells in vitro, bone marrow-derived macrophages and splenic T cells were harvested from wild-type C57Bl / 6 mice. Macrophages were polarized with cytokines and differentiated into M1 or M2, or left untreated. Splenic T cells were sorted into three major T cell populations: CD8+, CD4+CD25-, and CD4+CD25+ (Treg). All populations were treated with a low dose of RT (0.5 Gy × 4) one day after polarization or sorting. Because immune cells are radiosensitive, treatment did not include an ablative dose. Characterization of T cell populations 6 hours after the last dose revealed a significant decrease in Treg viability, but this was not observed in the CD8 or CD4+CD25- T cell populations (see Figure 9D). Within the sorted Treg population, cells treated with low-dose RT showed a trend toward decreased CD25 surface expression and a significant decrease in FOXP3 and CD25 dual expression, as shown in Figure 9E. M2-polarized macrophages treated with low-dose RT showed a significant decrease in the expression of the M2 marker CD206, as shown in Figure 9F. M2-polarized macrophages also tended to express only small amounts of IL-10 after RT, indicating repolarization after treatment (see Figure 9G). These data demonstrate that PAM-RT enhances the cytotoxicity of ablative RT without inhibiting its immunomodulatory effects. Furthermore, our low-dose RT reduces Tregs and reprograms M2 macrophages to a reduced immunosuppressive phenotype.

[0076] Local PAM-RT promotes remodeling of the tumor microenvironment by reducing Treg and M2 macrophages To investigate the immunological consequences of PAM-RT on the tumor microenvironment (TME), irradiated 3LL tumors were harvested on days 6 and 10 after the initiation of RT, as shown in Figure 10A. A significant increase in leukocyte infiltration was observed in tumors treated with PAM-RT compared to ablative RT alone. Phenotyping of infiltrated leukocytes revealed a significant decrease in intratumoral Tregs (see Figure 10B) and a significant increase in the CD8 / Treg ratio in mice treated with PAM-RT compared to ablative RT alone at day 6. As seen in Figure 10C, RT-PCR on whole tumor RNA demonstrated a significant decrease in FOXP3 mRNA expression in tumors treated with PAM-RT at day 6. As seen in Figure 10D, this reconstitution occurred along with a trend toward increased intratumoral CD8+ T cells secreting granzyme B at days 6 and 10, indicating an enhanced effector CTL response. Characterization of intratumor myeloid cell populations revealed a significant decrease in IL-10-secreting macrophages and a slight decrease in CD206 expression at day 6, as well as a trend toward decreased IL-10 secretion and a significant decrease in CD206 expression at day 10 after the initiation of treatment, as shown in Figure 10E. In summary, PAM-RT promotes TME reconstitution by decreasing immunosuppressive Treg and M2 macrophages while increasing CTL activity.

[0077] Local PAM-RT enhances systemic T responses and reduces Tregs To examine immune cells in secondary lymphoid organs, tumor-draining lymph nodes and spleens were harvested on days 6 and 10 after the initiation of RT, as in previous studies (e.g., Figure 10A). As seen in Figures 11A and 11B, mice treated with PAM-RT exhibited a significant increase in leukocytes in the spleen and draining lymph nodes on days 6 and 10. For tumor-draining lymph nodes (TDLNs), mice treated with PAM-RT exhibited a significant increase in CD8+ cells and a trend toward a decrease in Tregs on both days 6 and 10, as seen in Figure 12A. Analysis of spleens revealed minimal changes in CD8+ T cells and a significant decrease in Tregs on day 6 in mice treated with PAM-RT compared with the single ablation dose, which reversed on day 10, revealing a significant increase in CD8+ T cells and minimal changes in Tregs (Figure 12B). Examination of the functional status of splenic T cells on days 6 and 10 using ELISPOT revealed a trend toward increased granzyme B-secreting effector cells (Fig. 12C) and a significant increase in IFNγ-secreting effector cells (Fig. 12D) in PAM-RT-treated mice on day 6. Analysis on day 10 revealed a significant increase in granzyme B-secreting effector cells and a trend toward increased IFNγ-secreting effector cells. Although treatment in these mice was localized to the primary tumor, it resulted in systemic modulation, similar to the trends occurring in the tumor, reducing immunosuppression and enhancing T cell responses.

[0078] Systemic PAM-RT delays metastatic progression and increases survival To investigate whether PAM-RT directed at organs prone to metastasis could suppress progression after a series of hypofractionated ablative RT directed at the primary tumor in a low-immunogenic, highly metastatic cancer, we administered a series of local PAM-RT (22 Gy + 0.5 Gy × 4) to orthotopic 4T1 breast cancer in Balb / c mice. PAM-RT directed at the primary tumor tended to delay local tumor progression, but did not increase survival, and all treated mice succumbed to metastatic disease (Figure 13A-C). Given that local treatment with PAM-RT remodeled the TME, we transitioned the use of PAM-RT to treat metastatic organs after primary tumor ablation. To ensure sufficient local tumor control, Balb / c mice bearing palpable 4T1 tumors were treated with three 20 Gy doses directed at the primary tumor over three consecutive days. As expected, ablation of the primary tumor alone did not rescue the animals from lung metastases. Despite the induction of antitumor immune responses after RT to the primary tumor, RT-induced CTLs may have been excluded from infiltrating metastatic sites. To test this, we treated whole lungs, an organ prone to metastasis, with daily doses of 0.5 Gy for 12 days, starting 4 days after completion of RT to the primary tumor (Figure 14A). As shown in Figure 14B, survival rates were significantly increased in these animals after PAM-RT to the whole lung compared with RT to the primary tumor alone. Examination of the metastatic burden in treated mice revealed fewer metastatic foci in PAM-RT-treated lungs by macroscopic examination after India ink injection, as seen in Figure 14C, and in histological specimens in Figure 14D. PET scans of 4T1 mice showed similar results to those from lung samples, with lungs receiving PAM-RT having a smaller metastatic burden (Figures 15A and B). These data indicate that PAM-RT can be administered either directly to the primary tumor at a dose of 0.5 Gy × 4 for local control, or, when treating systemic disease, with delayed administration of PAM-RT to organs prone to metastases, slowing tumor progression and increasing survival.

[0079] Systemic PAM-RT remodels the metastatic niche and reduces lung Tregs Characterization of immune cells in PAM-RT-treated lungs demonstrated a reduction in the immunosuppressive phenotype of these cells, a result similar to that observed with localized PAM-RT treatment. As seen in Figure 16A, there was a significant reduction in Tregs in whole lungs treated with PAM-RT, leading to a significant decrease in the CD8 / Treg ratio (Figure 16B). Figure 16B illustrates the whole lung phenotype by flow cytometry 19 days after primary tumor ablation (Figure 16C). Phenotyping of lung T cells revealed a significant increase in GzB secretion by both CD8+ and CD4+ T cells (Figure 16C). Histological staining of micrometastases in PAM-RT-treated lungs revealed a massive infiltration of CD8+ T cells, while FoxP3+ cells were reduced, compared to lesions in mice that only underwent primary tumor ablation (Figure 16D). Untreated animals had splenic metastases accompanied by splenic enlargement and a decrease in CD45+ leukocytes. RT to the primary tumor and RT to the primary tumor plus PAM-RT to the lungs reduced spleen size, significantly increased CD45+ leukocytes (Figure 16E), and increased CD3+ T cell numbers (Figure 16F), whereas RT plus PAM-RT to the lungs restored spleen numbers to baseline levels seen in tumor-free wild-type animals. The expansion of peripheral myeloid cells was associated with G-CSF-secreting 4T1 tumors, consistent with previous reports. There was a massive increase in CD45+ macrophages among spleens of 4T1 tumor-bearing mice, and a progressive decrease in these cells in mice treated with ablation alone versus ablation plus PAM-RT. Interestingly, in untreated tumor-bearing animals, splenic macrophages were immature and had reduced MHC class II expression. Treatment with PAM-RT for lung ablation + / - for primary tumors significantly increased class II MHC expression in macrophages, indicating that PAM-RT treatment activated macrophages ( Figure 16G ).These experiments demonstrate that PAM-RT treatment can promote TME remodeling in primary tumors as well as metastatic sites by reducing Tregs, activating macrophages to a proinflammatory phenotype, and promoting infiltration of CD8+ CTLs in metastatic tumors.

[0080] material and method Cell lines and mice The mouse Lewis lung carcinoma cell line, 3LL, was purchased from ATCC and grown in supplemented DMEM (10% FBS, 5% sodium pyruvate, 2.5% NEAA, 1% antibiotic / antimitotic agent). The mouse mammary carcinoma cell line, 4T1, was purchased from ATCC and grown in supplemented DMEM (10% FBS, 1% antibiotic / antimitotic agent). Cell lines were used between passages 4 and 8 and tested for mycoplasma every 4 months using MycoAlert (Lonza LT07-705). Six- to eight-week-old C57BL / 6 mice and 10- to 12-week-old BalB / C mice were ordered from NCI, and athymic nude mice were ordered from Charles River. All studies performed were approved by the Institutional Animal Care and Use Committee.

[0081] In vivo tumor studies C57BL / 6 mice were challenged subcutaneously with 1.5 x 10 3LL cells, and BalB / C mice were challenged orthotopically with 2 x 10 4T1 cells in the mammary fat pad. 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 ellipsoid formula: V = (π / 6 x depth x width x height).

[0082] CT-guided radiotherapy for tumor-bearing mice Radiation is administered using Xstrahl Limited's Small Animal Radiation Research Platform (SARRP). Image-guided radiation therapy is performed using the SARRP's built-in cone beam CT (CBCT). After CBCT acquisition, treatment plans are constructed using Muriplan.

[0083] Tumor and immune cell analysis Tumor cells were cultured, seeded, and treated using specific irradiation schemes. Cells were harvested 6 and 24 hours after the final treatment on day 5. Cells were then collected and stained for flow cytometry. Bone marrow-derived macrophages were polarized into M1 (LPS 100 ng / mL + IFNg 50 ng / mL) and M2 (IL-4 10 ng / mL) 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 allowed to rest overnight before radiation treatment. Immune cells were harvested 6 hours after the final radiation dose.

[0084] Tumor treatment Tumors or whole lungs were harvested on ice, weighed, and washed. After manual removal with a razor blade, tumors or whole lungs were transferred to 1 mL of digestion buffer (10% FBS, collagenase I and IV (100 μg / mL) (Sigma) and 1x DNase I (Thermo Scientific)) in a 15 mL conical tube with a magnetic stir bar. The tube was then incubated at 37°C for 15 minutes while rotating, and then transferred to a stir plate for 15 minutes of manual digestion. The single cell suspension was filtered. The cells were resuspended for flow cytometry.

[0085] Flow cytometry analysis Cells were stained with surface-staining antibodies for 30 minutes at 4°C. Antibodies used included CD45, CD3, CD4, FOXP3, CD8, GzB, CD11b, MHCII, IL-10, TNFα, and CD206. After washing, cells were either fixed with 4% PFA or permeabilized for intracellular staining with BD Pharminogen Transcription Factor Buffer Set according to manufacturer's instructions. Intracellular staining was performed for FOXP3, TNFα, IL-10, and granzyme B, followed by fixation with 4% PFA. Cells were collected on an LSRII flow cytometer (BD Biosciences) and analyzed using Flow Jo software (Tree Star).

[0086] Collected from lymphoid organs for immunological analysis Spleens and draining lymph nodes were harvested on ice and processed for single-cell suspension. In spleens, red blood cells were lysed using ACK lysis buffer (Lonza). Cells were counted before resuspension in complete RPMI (10% heat-inactivated FBS, 1% antibiotic / antimitotic). For intracellular cytokine analysis, GolgiStop and monensin were added for 3 hours at 37°C. Single-cell suspensions were further processed for flow cytometry analysis, as with tumors.

[0087] ELISOPT assay For immunoassays, processed spleens were seeded at 1x10^6 cells per well onto coated ELISPOT plates and incubated overnight before further processing. IFNγ ELISPOT reagents were ordered from BD biosciences and the manufacturer's protocol was followed. Granzyme B ELISPOT reagents were ordered from R&D and the manufacturer's protocol was followed.

[0088] Tumor tissue staining Tumors and lungs were harvested, transferred to 4% PFA or 10% formalin, and stored at 4°C. Samples were then transferred to 70% and embedded in paraffin. Blocks were sectioned and double-stained for CD8 and FOXP3.

[0089] statistical analysis Statistical analysis was performed using PRISM 7 (Graphpad Software) software. Analysis was performed by multiple-group comparison using the Student's t-test or ANOVA analysis. Data are representative means ± standard deviations. Survival curves were analyzed using the log-rank (Mantel-Cox) test and the Gehan-Wilcoxon-Breslow test. For statistical significance, P values ​​are expressed as *p<0.05, **p<0.005, ***p<0.0005, and ****p<0.0001.

[0090] RT-PCR analysis of TME Six days after the start of treatment, tumors were harvested on ice and weighed. Tumors were manually excised on ice and then 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 using a Nanodrop with a 260nm / 280nm ratio of approximately 2. cDNA synthesis was achieved using a Verso cDNA synthesis kit (Thermo Fisher) according to the kit's protocol. For RT-PCR in 384-well plates on an ABI 7900HT, a Powerup SYBR Green kit (Thermo Fisher) was used. The primers used were FOXP3 F-ACTCGCATGTTCGCCTACTTCAG and R-GGCGGATGGCATTCTTCCAGGT, and β2-microglobulin F-TTCTGGTGCTTGTCTCACTGA and R-CAGTATGTTCGGCTTCCCATTC. ΔCT was calculated using SDS 2.4 and compared with the untreated control group.

[0091] PET imaging Mice were fasted overnight and transported to the imaging facility. They were injected with 300–400 μCi (12–15 MBq) of [18F]fluoro-2-deoxyglucose (FDG) in 0.1 ml of saline via the tail vein, and imaging began 1 h after injection. Mice were anesthetized with isoflorane and imaged on an Inveon Multimodality scanner (Siemens). Analysis was performed using either ASIPRO or IRW (both Siemens) dedicated software.

[0092] Lungs treated with India ink Treated 4T1 tumor-bearing mice were sacrificed. Lungs were inflated intratracheally with 10% India ink. Lungs were harvested and washed with 1 L of water before being preserved in Feket's solution (70% EtOH (300 ml), 37% formaldehyde (30 ml), glacial acetic acid (5 ml)) to allow decolorization of macrometastases. Metastatic foci were enumerated by counting.

[0093] Example 2 Quantitative and qualitative T cell analysis after radiation therapy To test the radiation therapy scheme, 6-week-old C57BL / 6 mice were inoculated subcutaneously with 3LL in the dorsal foot. Before treatment, tumors were 50 mm 3 Tumor volumes were calculated using the ellipsoid formula: V = (π / 6 x depth x width x height).

[0094] The inoculated mice were divided into four treatment groups. Group 1 received no treatment; Group 2 received a single dose of 24 Gy; Group 3 received a single dose of 22 Gy followed one day by four doses of 0.5 Gy spaced one day apart; and Group 4 received four doses of 0.5 Gy spaced one day apart followed one day by a single dose of 22 Gy. Tumor size was measured three times weekly, and survival was assessed daily. As shown in Figure 18A, mice in Group 3 (c) had a better survival outcome than mice in Groups 1, 2, and 4. Some mice in Group 3 survived for nearly three months, while all mice in Groups 1 and 2 and most mice in Group 4 died after approximately one month. Another group of mice was treated as above and injected with FITC-dextran 6 days after the first treatment, and tumor vasculature was quantified. As shown in Figure 18B, mice in groups 3 and 4 showed increased vascular density compared to mice in groups 1 and 2.

[0095] To investigate possible immune effects, further experiments were conducted using immunocompromised C57B16 mice and nude mice. Mice were divided into three groups and treated similarly to groups 1, 2, and 3 above. In C57B16 mice, tumor growth in group 3 was slower than in groups 1 and 2, as seen in Figure 19A. This effect was lost in nude mice, as shown in Figure 19B. Similarly, comparing Figures 20A and 20B, the survival benefit seen in C57B16 mice was not observed in nude mice.

[0096] To investigate the effect of radiation therapy on immune responses, three different tumor cell lines were used, which consisted of 1 × 10 5 3LL, 2×10 5 UN-KC-6141, and 4 × 10 5The MOE HPV E6 / 7 / H-Ras cells were used. As described above, C57Bl6 mice were inoculated with the cells, and treated mice of each cell type were divided into six treatment groups: (1) no treatment, (2) 24 Gy, (3) 20 Gy 1 day followed by four doses of 1 Gy per day, (4) 22 Gy 1 day followed by four doses of 0.5 Gy per day, (5) four doses of 1 Gy per day, and (6) four doses of 0.5 Gy per day. Six days after the first treatment, tumors were harvested and analyzed for leukocyte infiltration by flow cytometry, as described below. For all cell lines tested, increased leukocyte infiltration was observed in the third group compared to the other groups (see Figures 21A-C). In mice treated with H-Ras cells, group 4 was more effective than groups 1, 2, 5, and 6 (Figure 21B), but not in mice treated with 3LL or 4T1 tumor cells.

[0097] To determine whether the above irradiation scheme would be effective for combination therapy, mice were injected with tumor cells as described above and treated with trabectedin alone, 24 Gy and trabectedin, or 22 Gy 1 day followed by three weekly doses of 0.5 Gy and three weekly doses of trabectedin. Survival and tumor growth were recorded for each group. Figure 22A shows the survival rates of mice in the different groups, with the highest survival rate observed in the group treated with 22 Gy 1 day followed by four daily doses of 0.5 Gy and trabectedin. Figures 22B–D show the growth curves of the different tumors within each treatment group, with the slowest growth rate observed in the group treated with 22 Gy 1 day followed by four daily doses of 0.5 Gy and trabectedin (Figure 22D).

[0098] Further combination experiments were performed using mice inoculated with 4T1 cells into the fourth mammary fat pad. Approximately 8 days later, the mice were treated as follows: 1.3 doses of 20 Gy, 2. Three doses of 20 Gy and anti-PD1 therapy; 3. Three 20 Gy doses followed 12 days later by four daily 0.5 Gy doses (administered to the whole lung), or 4. Three doses of 20 Gy and anti-PD1 therapy followed 12 days later by four daily doses of 0.5 Gy (given to the whole lung) and anti-PD1 therapy. The treatment regimen is illustrated in Figure 23. As shown in Figures 24A and 24B (excluding mice with local failure of the primary tumor, which may worsen survival), mice treated in group 4 had the longest survival rates. Mice treated with 4T1 cells intravenously and either anti-PD1 therapeutic agent alone, four daily doses of 0.5 Gy alone, or anti-PD1 therapeutic agent and four daily doses of 0.5 Gy had similar survival rates to untreated mice (see Figure 25).

[0099] method Cell lines and mice The mouse Lewis lung carcinoma cell line, 3LL, was purchased from ATCC and grown in supplemented DMEM (10% FBS, 5% sodium pyruvate, 2.5% NEAA, 1% Pen / Strep). 4T1 mouse mammary carcinoma was purchased from ATCC and grown in supplemented DMEM (10% FBS, 1% Pen / Strep).

[0100] Six-week-old C57BL / 6 mice were ordered from NCI and maintained under pathogen-free conditions.

[0101] CT-guided radiation therapy for tumor-bearing mice Radiation will be administered using Xstrahl Limited's Small Animal Radiation Research Platform (SARRP). Image-guided radiation therapy will be performed using the SARRP's built-in cone beam CT (CBCT). After CBCT acquisition, treatment plans will be constructed using Muriplan.

[0102] Immunotherapy treatment of tumor-bearing mice Tumor-bearing mice randomized to the immunotherapy treatment group received PD-1 (200 μg), αCD40, 4-1BBL, and GM-CSF intraperitoneally (ip) every 3 days for a total of 5 doses, with or without antigen vaccine.

[0103] Syntac administration To induce activation of antigen-specific effector CD8+ T cells, we use a Syntac construct that conjugates the T cell costimulatory domain 41BBL to a tumor-type-specific antigen. Tumor-bearing mice randomized to Syntac receive a single 300µg intraperitoneal treatment. Mice receiving antigen-specific Syntac treatment are compared with mice treated with irrelevant Syntac or with the costimulatory domain 41BBL alone.

[0104] Tumor digestion Tumors were harvested on ice and weighed. After manual dissection into 1 mm x 1 mm sections using a razor blade, tumors were transferred to 1 mL of digestion buffer, 5% FBS, collagenase I and IV (100 u / mL) (Sigma), and 1xDNase I (Thermo Scientific) in a 15 mL conical tube equipped with a magnetic stir bar. The tube was incubated at 37°C for 30 minutes and then transferred to a stir plate for 30 minutes of manual digestion. The single-cell suspension was filtered and resuspended for analysis.

[0105] Flow cytometry analysis of immune cells Single-cell suspension cells were stained with surface-staining antibodies for 30 minutes at 4°C. After washing, cells were either fixed with 4% PFA or permeabilized for intracellular staining with BD Pharminogen Transcription Factor Buffer Set according to manufacturer's instructions. Intracellular staining was for FOXP3 and Ki-67, followed by fixation with 4% PFA. Cells were collected on an LSRII flow cytometer (BD Biosciences) and analyzed using Flow Jo software (Tree Star).

[0106] antibody 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).

[0107] Flow cytometry antibodies: CD45, CD3, CD4, CD8, CD69, PD1, CD11b, Gr1, Ly6C, LY6G, CD11c, NK1.1, NKG2D, MHC II(IA), and F4 / 80.

[0108] Tumor microenvironment analysis Six days after treatment, tumors were harvested on ice and weighed. While still on ice, tumors were transferred to 1.7 mL Eppendorf tubes and homogenized in 4.5 μL of PBS with 1× protease and phosphatase inhibitors (cell signaling) per milligram of tissue. The tubes were spun down at 14,000 g for 15 minutes. The supernatant was transferred to a clean tube and stored at -80°C.

[0109] Quantification of vascular density using FITC-dextran Mice were injected subcutaneously in the flank with 1 x 10^5 3LL. Tumors were allowed to grow for approximately 14 days until they reached approximately 6 mm in size. Mice were randomized to receive no treatment, 24 Gy on day 1, 22 Gy + 0.5 Gy x 4 starting on day 2, or 0.5 Gy x 4 starting on day 2. Six days after the start of treatment, 70 kDa FITC-dextran (25 mg / mL) was intravenously injected and allowed to circulate 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 spun down at 14,000 rpm for 5 minutes. Supernatants were collected and run at 485 / 535 for fluorescence. Fluorescence per mg was then calculated. Blood serum was used as an injection control.

[0110] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be utilized in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A radiation therapy system, comprising: a radiation source that provides an ablation dose and a sub-ablative dose; a processor coupled to the radiation source, the processor configured with instructions to deliver an ablation dose of radiation therapy to a first region and, after the ablation dose, deliver a sub-ablative dose of radiation therapy to a second region; 1. A radiation therapy system comprising:

2. A radiation therapy system as described in claim 1, including an adjustment device configured to adjust the amount and direction of the radiation beam.

3. A radiation therapy system as described in claim 1 or 2, wherein the sub-ablation dose is administered at least one hour after the ablation dose.

4. A radiation therapy system described in any one of claims 1 to 3, wherein the sub-ablation dose is administered at least one day after the ablation dose.

5. A radiation therapy system described in any one of claims 1 to 3, wherein the sub-ablation dose is administered at least 12 days after the ablation dose.

6. A radiation therapy system described in any one of claims 1 to 5, wherein the sub-ablation dose is administered more than once.

7. A radiation therapy system as described in claim 6, wherein the second sub-ablation dose is administered one day after the first sub-ablation dose.

8. The radiation therapy system of claim 6, wherein the ablation dose is administered over at least two, three, or four consecutive days.

9. A radiation therapy system as described in claim 8, wherein the second sub-ablation dose is administered one week after the first sub-ablation dose.

10. A radiation therapy system described in any one of claims 1 to 9, wherein the sub-ablation dose is between 0.1 Gy and 2 Gy.

11. A radiation therapy system as described in claim 10, wherein the sub-ablation dose is 0.5 Gy.

12. A radiation therapy system as described in claim 10, wherein the sub-ablation dose is 1 Gy.

13. A radiation therapy system described in any one of claims 1 to 12, wherein the ablation dose is between 20 Gy and 100 Gy.

14. A radiation therapy system as described in claim 13, wherein the ablation dose is 20 Gy.

15. A radiation therapy system described in any one of claims 1 to 14, wherein the first region includes a primary tumor.

16. The radiation therapy system of claim 15, wherein the second region includes a primary tumor.

17. The radiation therapy system of claim 15, wherein the second region includes a metastatic tumor.

18. A radiation therapy system described in any one of claims 1 to 14, wherein the first region includes a metastatic tumor.

19. The radiation therapy system of claim 18, wherein the second region includes a primary tumor.

20. The radiation therapy system of claim 18, wherein the second region includes a metastatic tumor.

21. A radiation therapy system described in any one of claims 1 to 20, wherein the first region includes an area different from the second region.

22. A radiation therapy system described in any one of claims 1 to 20, wherein the first region includes the same area as the second region.

23. The radiation therapy system of any one of claims 15 to 17 and 19, wherein the primary tumor comprises a tumor of the breast, bladder, brain, colon, rectum, endometrium, kidney, pancreas, prostate, liver, lung, skin, thyroid, uterus, lymph node, tonsil, thymus, spleen or bone marrow, or a combination thereof.

24. A radiation therapy system described in any one of claims 17 to 20, wherein the metastatic tumor comprises a tumor of the bone, lymph node, lung, liver, brain, adrenal gland, breast, eye, kidney, muscle, pancreas, salivary gland or spleen, or a combination thereof.

25. A radiation therapy system described in any one of claims 1 to 24, wherein administering the ablation dose and the sub-ablation dose increases the infiltration of effector T cells into the first region, the second region, or both.

26. A radiation therapy system described in any one of claims 1 to 25, wherein administering the ablation dose and the sub-ablation dose reduces the influx of immunosuppressive regulatory T cells into the first region, the second region, or both.

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