Transient cell-in-cell formation underlies tumor relapse and resistance to immunotherapy
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Evaluation Summary:
Although immunotherapy has revolutionized the cancer field, most tumors do not respond, and in those that do respond, acquired resistance is often inevitable. Several mechanisms have been proposed to be involved in acquired resistance to immunotherapy. In the present study, the authors show that tumor cells from multi-cellular structures protect the inner core of tumor cells via the prevention of penetration by lytic molecules. The formation of these structures is mediated by anti-tumor T cells even with tumors that have retained their immunogenic neoantigens. This work identifies a novel possible resistance mechanism to immune-mediated tumor killing.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Despite the remarkable successes of cancer immunotherapies, the majority of patients will experience only partial response followed by relapse of resistant tumors. While treatment resistance has frequently been attributed to clonal selection and immunoediting, comparisons of paired primary and relapsed tumors in melanoma and breast cancers indicate that they share the majority of clones. Here, we demonstrate in both mouse models and clinical human samples that tumor cells evade immunotherapy by generating unique transient cell-in-cell structures, which are resistant to killing by T cells and chemotherapies. While the outer cells in this cell-in-cell formation are often killed by reactive T cells, the inner cells remain intact and disseminate into single tumor cells once T cells are no longer present. This formation is mediated predominantly by IFNγ-activated T cells, which subsequently induce phosphorylation of the transcription factors signal transducer and activator of transcription 3 (STAT3) and early growth response-1 (EGR-1) in tumor cells. Indeed, inhibiting these factors prior to immunotherapy significantly improves its therapeutic efficacy. Overall, this work highlights a currently insurmountable limitation of immunotherapy and reveals a previously unknown resistance mechanism which enables tumor cells to survive immune-mediated killing without altering their immunogenicity.
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Evaluation Summary:
Although immunotherapy has revolutionized the cancer field, most tumors do not respond, and in those that do respond, acquired resistance is often inevitable. Several mechanisms have been proposed to be involved in acquired resistance to immunotherapy. In the present study, the authors show that tumor cells from multi-cellular structures protect the inner core of tumor cells via the prevention of penetration by lytic molecules. The formation of these structures is mediated by anti-tumor T cells even with tumors that have retained their immunogenic neoantigens. This work identifies a novel possible resistance mechanism to immune-mediated tumor killing.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to …
Evaluation Summary:
Although immunotherapy has revolutionized the cancer field, most tumors do not respond, and in those that do respond, acquired resistance is often inevitable. Several mechanisms have been proposed to be involved in acquired resistance to immunotherapy. In the present study, the authors show that tumor cells from multi-cellular structures protect the inner core of tumor cells via the prevention of penetration by lytic molecules. The formation of these structures is mediated by anti-tumor T cells even with tumors that have retained their immunogenic neoantigens. This work identifies a novel possible resistance mechanism to immune-mediated tumor killing.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In the present study, the authors investigate mechanisms of tumor cell resistance to T cell-mediated killing. They show that, in the presence of tumor-specific T cells, tumor cells form multi-cellular structures that protect the inner core of tumor cells via the prevention of penetration by lytic molecules. When these T cells are absent or removed, the tumor cells return to their single cells. Although neoantigen 'loss' has been shown as an immunotherapy escape mechanism, this study also shows in robust fashion that relapsed tumor cells share most neoantigens with their primary tumors. Overall this is a very thorough and rigorous study and the data firmly support the conclusions.
Strengths
1. Thorough and rigorous investigation of the hypotheses.
2. Data firmly support the conclusions.
3. Possible new …Reviewer #1 (Public Review):
In the present study, the authors investigate mechanisms of tumor cell resistance to T cell-mediated killing. They show that, in the presence of tumor-specific T cells, tumor cells form multi-cellular structures that protect the inner core of tumor cells via the prevention of penetration by lytic molecules. When these T cells are absent or removed, the tumor cells return to their single cells. Although neoantigen 'loss' has been shown as an immunotherapy escape mechanism, this study also shows in robust fashion that relapsed tumor cells share most neoantigens with their primary tumors. Overall this is a very thorough and rigorous study and the data firmly support the conclusions.
Strengths
1. Thorough and rigorous investigation of the hypotheses.
2. Data firmly support the conclusions.
3. Possible new mechanism of resistance to T cell-mediated tumor killing.Weaknesses
1. Relevance to current immunotherapy treatment regimens is weak.
2. Figure legends overall need work. -
Reviewer #2 (Public Review):
The current manuscript explains a novel phenomenon observed in cancer cells exposed to immune therapy, named cell-in-cell contact where several tumor cells join together without being fused to form these unique structures. The authors suggest this phenomenon as a mechanism for therapy resistance and see it often in relapsed tumors (melanoma and breast cancer). The authors followed animals that developed resistance to melanoma immunotherapy (TNF-a+CD40L+TRP1 melanoma antigen). Despite all mice almost clearing the tumor, 50% of these animals relapsed 10 days later. A similar regrowth pattern was noticed in breast cancer treated with allogeneic antibodies and DC adjuvant. Also, treating melanoma-bearing mice with splenic CD8+ T cells expressing TCR against gp100, or TRP2 melanoma antigens induced significant …
Reviewer #2 (Public Review):
The current manuscript explains a novel phenomenon observed in cancer cells exposed to immune therapy, named cell-in-cell contact where several tumor cells join together without being fused to form these unique structures. The authors suggest this phenomenon as a mechanism for therapy resistance and see it often in relapsed tumors (melanoma and breast cancer). The authors followed animals that developed resistance to melanoma immunotherapy (TNF-a+CD40L+TRP1 melanoma antigen). Despite all mice almost clearing the tumor, 50% of these animals relapsed 10 days later. A similar regrowth pattern was noticed in breast cancer treated with allogeneic antibodies and DC adjuvant. Also, treating melanoma-bearing mice with splenic CD8+ T cells expressing TCR against gp100, or TRP2 melanoma antigens induced significant tumor regression, followed by tumor recurrence in all treated mice. These last ones were resistant to treatment. Four melanoma cell lines were established from therapy relapse. Tumor cells kept expressing tumor antigene (gp100, TRP2, and MHC-I. Whole exome analysis (WES) indicated a comparable neoantigen burden across all samples. The neoantigen burden analysis of 2 data sets, in relapsed melanoma and in non-small cell carcinoma patients, showed the majority of neoantigens were shared between relapsed tumors and their corresponding primary tumor.
The authors established cell lines from relapsed tumors following treatment with gp100-reactive T cells and assessed their responsiveness to treatment with gp100-reactive T cells. Upon exposure to T cells, cancer cells cluster together, several nuclei surrounded by a single membrane and cortical actin, pathologists indicated that about half the cells that survived immunotherapy formed these structures. Using fluorescent tagging showed that there is no cell fusion and cells that formed cell-in-cell structure, went back to single cell status after removing the treatment.
By coculturing tumor cells with CD8 and to less extent CD4 induced the same phenomenon in vitro. It is worthy to note that Nude SCID- gamma-/- (NSG) mice didn't have such structures. It is important to note that immortalized mammary epithelial cells were completely killed by allogeneic T cells and did not form a cell-in-cell structure, thus these structure seems more cancer-specific. Also, not all cancer lines formed cell-in-cell structures, including pancreatic tumors and B cell lymphoma, while almost all the colon and ovarian carcinoma tumor cells that survived T cell killing, were organized in such structures. Furthermore, histology analysis of T cell and myeloid lymphomas, as well as glioblastomas, did not show a trace of such structures. The authors showed the phenomenon in primary melanoma too.
Similar to activated T cells, secreted granules isolated from T cells were sufficient to induce cell-in-cell formation. Secreted granules were specific to activated T cells, but not activated NK, macrophages, and immortalized melanocytes. This cluster formation seems to be mainly INF-g dependent which in its role phosphorylate activator of transcription 3 (STAT3) and early growth response-1 (EGR-1) in the tumors cells. Also, they found that IFNγ -stimulated NK cells and macrophages, but not B cells could induce a tumor cell-in-cell formation, at a lower percentage compared to activated T cells.
The authors investigated the effect of cell-in-cell formation in protecting tumor cells from T-cell-mediated killing. They noticed that the outer layer is lysed by T cells whereas the inner layer stays safe and intact. Immunostaining of T cells attacking cell-in-cell tumors, showed that the distribution of granzyme B and perforin is almost limited to the outer cell. While these interactions were sufficient to kill single tumor cells, they were insufficient to induce caspase 3/7 activity in the inner cell, leaving it intact
and alive.The author concluded that tumor cells that survive chemotherapy and immunotherapy form unique cluster structures and generate membrane architecture impenetrable by immune-derived lytic granules, cytotoxic compounds, and chemotherapies leading to therapy resistance.
The topic is novel and interesting, the manuscript is well written! We do believe the authors have included enough in vivo and imaging to support their findings.
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