Functional tumor cell-intrinsic STING, not host STING, drives local and systemic antitumor immunity and therapy efficacy following cryoablation

CD8+IFN-γ+ T cells are an effector population shown to be a target of downstream STING signaling involving the IFN-β-CXCL9/10 axis.

“an effector population shown to be a target of downstream STING signaling involving the IFN-β-CXCL9/10 axis.54–58”

• cites [54] sourcePartly supported

  The sections “Regulation of the STING pathway in APCs and its role in cancer” and “Type I IFNs in the generation of antitumor immune responses” link STING activation to IFN-β production and antitumor CD8+ T-cell responses. The latter section also associates CXCL9 and CXCL10 with T-cell recruitment, but our copy does not establish the stated IFN-β–CXCL9/10 signaling axis or identify CD8+IFN-γ+ T cells specifically as its target. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [55] sourcePartly supported

  The Results section (page unavailable) shows that CXCL9 and CXCL10 recruit human CD8+ effector T cells in migration assays. It does not identify those cells as IFN-γ+ or establish that their recruitment is downstream of STING signaling through IFN-β. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [56] Vonderhaar, 2021, STING activated tumor-intrinsic type I interferon signaling promotes Cxcr3 dependent antitumor immunity in Pancreatic cancer, Cell Mol Gastroenterol Hepatol sourcePartly supported

  The Results section reports that STING agonist increased IFN-β expression and the frequency of CXCR3+CD8+ T cells in pancreatic tumor models. The Discussion links STING activation, type I interferon signaling, and CXCL9/CXCL10 to effector T-cell infiltration. Our copy does not establish that the affected CD8+ cells were specifically IFN-γ+. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [57] sourcePartly supported

  The Results section reports reduced T-cell infiltration in STING-deficient tumors and links effector T-cell migration to CXCL9/10-producing dendritic cells; it also describes STING-dependent IFN-β and CXCL9/10 production. The paper supports this pathway for effector CD8+ T-cell recruitment, but does not establish that the recruited CD8+ cells are specifically IFN-γ-positive. Its Discussion separately identifies IFN-γ-producing CD4+ Th1 cells. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [58] Reschke R , Olson DJ . Leveraging STING, Batf3 Dendritic cells, Cxcr3 ligands, and other components related to innate immunity to induce A "hot. Cancers (Basel 2022;14(10. sourcePartly supported

  Section 3.1 (page unavailable) states that STING-induced CXCL9/10 expression depends on type I interferons, including IFN-β. Section 3 (page unavailable) says CXCL9/10 promote T-cell migration and are associated with CD8+ effector T cells. The paper does not specifically identify CD8+IFN-γ+ T cells as a demonstrated target of that signaling axis. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Clinical outcomes of tumor cryoablation-both obtaining local control and achieving an abscopal effect-are highly unpredictable with variable magnitudes of response and frequent failure of local recurrence.

“Despite these theoretical benefits, clinical outcomes of tumor cryoablation—both obtaining local control and achieving an abscopal effects—are highly unpredictable with variable magnitudes of response and frequent failure of local recurrence.”

• cites [76] Aarts, 2019, Cryoablation and Immunotherapy: an overview of evidence on its synergy, Insights Imaging sourcePartly supported

  The Abstract and Introduction say the abscopal effect is rarely observed, supporting that part of the claim. But the Conclusion says cryoablation has proven successful for local control in various cancers; it does not describe local control as highly unpredictable or report frequent local failure. The cited passages also do not establish variable magnitudes of clinical response. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [77] Yakkala, 2019, Cryoablation and Immunotherapy: an enthralling synergy to confront the tumors, Front Immunol sourcePartly supported

  The ‘Critical Aspects’ section (page unavailable) says some patients experience local tumor progression after cryoablation and that factors governing its effects remain ill-defined. The ‘Immunology of Cryoablation’ section (page unavailable) describes variable immune responses and limited abscopal regression. These passages support uncertainty in outcomes, but the source says only a fraction of patients have local progression, not that local failure is frequent. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [78] Baust, 2019, Cryoablation: physical and molecular basis with putative immunological consequences, Int J Hyperthermia sourcePartly supported

  The Cryo-immunological Responses section (page unavailable) says distant-tumor effects are not always observed, are transient, and vary in extent; the Introduction says freezing does not damage a targeted tumor uniformly. These passages support variable responses, but they do not establish that clinical local control is highly unpredictable or that local recurrence frequently occurs. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Gain-of-function STING-N153S mutation resulted in T cell deficiency.

“and gain-of-function STING-N153S mutation resulted in T cell deficiency.85”

• cites [85] sourceSupported

STING activity induces T cell apoptosis.

“as recent evidence demonstrated that STING activity induces T cell apoptosis,85–87”

• cites [85] sourceSupported
• cites [86] Gulen, 2017, Signalling strength determines Proapoptotic functions of STING, Nat Commun sourceSupported
• cites [87] sourcePartly supported

  The Results and Discussion section (“STING activation triggers T cell death”; page not provided) reports STING-dependent T-cell death in vitro and evidence implicating an apoptotic pathway. But it also suggests necroptosis can contribute and reports no T-cell death in its short-term in vivo experiment, so the unqualified claim that STING activity induces T-cell apoptosis overstates the finding. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Cryoablation of RMS 76-9STING-/- tumors in C57BL/6 mice was rendered completely ineffective, despite functionally intact STING signaling in the host.

“cryoablation of RMS 76-9STING-/- tumors in C57BL/6 mice was rendered completely ineffective, despite the presence of functionally intact STING signaling in the host (figure 4A).”

Removal of CD8+ T cells resulted in tumor growth that was not significantly different from untreated tumors.

“removal of CD8+ T cells resulted in tumor growth that was not significantly different from untreated tumors (figure 1D, online supplemental figure S1).”

RMS 76-9STING-/- cells exhibit faster growth kinetics in vitro compared with RMS 76-9STINGwt cells.

“Although RMS 76-9STING-/- exhibit faster growth kinetics in vitro as compared with RMS 76-9STINGwt (online supplemental figure S8A)”

• Also stated in: § Tumor-intrinsic STING mediates antitumor immune responses following cryoablation · plain

  “at baseline we saw a growth advantage of RMS 76-9STING-/- tumor as compared with RMS 76-9STINGwt tumor (online supplemental figure S8A) in vitro.”

A higher percent of MHC II and CD80 expressing DCs were found in cryoablated TME compared with non-cryoablated group.

“A higher percent of MHC II and CD80 expressing DCs were found in cryoablated TME as compared with those in the non-cryoablated group (figure 2B).”

On exposure to dead tumor lysate, Ifn-β transcription was upregulated only in live RMS 76-9STINGwt cells, while live RMS 76-9STING-/- cells remained unaffected.

“On exposure to dead tumor lysate, Ifn-β transcription was upregulated only in the live RMS 76-9STINGwt, while live RMS 76-9STING-/- cells remained unaffected (online supplemental figure S4E).”

Total IFN-β increased in both RMS 76-9STINGwt and M3-9-MSTINGwt TME compared with STING-/- tumor TME following cryoablation.

“Total IFN-β increased in both RMS 76-9STINGwt (figure 3F) and M3-9-MSTINGwt TME (figure 7A) as compared with the STING-/- tumor TME following cryoablation.”

A significant increase in RMS lung metastases was observed in C57BL/6 mice with cryoablated RMS 76-9STING-/- tumors.

“we also observed a significant increase in RMS lung metastases in the same group (figure 4B,C)”

Cryoablation of RMS 76-9 tumors significantly increases IFN-β production in the TME 3 days after cryoablation compared with non-ablated TME.

“we assessed and observed a significant increase in the amount of IFN-β production in the TME 3 days after cryoablation as compared with non-ablated TME (figure 3A).”

Both RMS 76-9STINGwt and RMS 76-9STING-/- cell lines exhibit similar tumor growth kinetics in C57BL/6 mice in the absence of cryoablation.

“both cell lines exhibit similar tumor growth kinetics in C57BL/6 mice in the absence of cryoablation (figure 4A).”

Two additional RMS 76-9STING-/- tumor clones, Clones 7 and 8, either alone or in combination, showed the same phenomenon of complete loss of antitumor efficacy following cryoablation as seen in figure 4A.

“we tested two additional RMS 76-9STING-/- tumor clones, Clones 7 and 8, either alone or in combination, and observed the same phenomenon of a complete loss of antitumor efficacy following cryoablation as seen in figure 4A, online supplemental figure S5).”

Cryotherapy increases Il-12β transcription in DCs isolated from cryoablated TME in vivo.

“Similarly, cryotherapy of RMS 76-9 increases Il-12β transcription by co-cultured BMDCs in vitro (figure 2C) as well as in DCs isolated from cryoablated TME in vivo (figure 2D).”

In DMXAA-treated RMS 76-9STING-/- cells, upregulation of IFN-β and phosphorylation of IRF3 were decreased compared with wild-type cells.

“Both the upregulation of IFN-β (online supplemental figure S4C) and the phosphorylation of IRF3 (online supplemental figure S4D) were decreased in DMXAA-treated RMS 76-9STING-/- as compared with wild-type cells.”

Cryoablation of STINGwt tumor dramatically increases a variety of chemokines and cytokines, including CXCL9 and CXCL10.

“Cryoablation of STINGwt tumor dramatically increases a variety of chemokines and cytokines, including the canonical chemokines, CXCL9 and CXCL10, as well as a variety of others (figure 7B–D).”

Cryoablated tumor TME harbored a significant increase in the frequency of CD45+ cells.

“Cryoablated tumor TME harbored a significant increase in the frequency of CD45+ cells (figure 2F).”

In contrast to wild-type mice, the significant changes in frequency of CD4+ T cells expressing IFN-γ or GZMB, and the frequency and intensity of GZMB-expressing CD8+ T cells were lost in STING-/- mice.

“In contrast to wild-type mice, the significant changes in frequency of CD4+ T cells expressing IFN-γ (figure 5G) or GZMB (figure 5I), and the frequency and intensity of GZMB-expressing CD8+ T cells (figure 5J,K) were lost.”

• AssumptionRests on an assumption: STING-/- mice lack functional host STING signaling.

No significant changes were observed in the myeloid cell populations within the cryoablated TME of either tumors.

“We did not observe any significant changes in the myeloid cell populations within the cryoablated TME of either tumors (figure 4O,P).”

Cryoablation of RMS 76-9STINGwt tumor in wild-type mice induced sustained local tumor control.

“cryoablation of RMS 76-9STINGwt tumor in wild-type mice not only induced sustained local tumor control (figure 4A)”

Cryoablated RMS decreased BMDM expression of CD206+ under similar co-culture conditions.

“cryoablated RMS also decreased bone marrow-derived macrophage (BMDM) expression of CD206+ under similar co-culture conditions (figure 2E).”

Cryotherapy of RMS 76-9 increases Il-12β transcription by co-cultured BMDCs in vitro.

“cryotherapy of RMS 76-9 increases Il-12β transcription by co-cultured BMDCs in vitro (figure 2C)”

Conventional flow cytometry confirmed a significant increase in IFN-γ-expressing CD45+/CD3+/CD8+ TILs and CD3+ TILs post cryoablation of RMS 76-9STINGwt tumor compared with RMS 76-9STING-/- tumor in STING-/- mice.

“Conventional flow cytometry analysis confirmed a significant increase in IFN-γ-expressing CD45+/CD3+/CD8+ TILs (figure 5E, F and H), and CD3+ TILs post cryoablation of RMS 76-9STINGwt tumor as compared with RMS 76-9STING-/- tumor in the setting of STING-/- mice”

• AssumptionRests on an assumption: STING-/- mice lack functional host STING signaling.

Depletion of CD4+ T cells resulted in partial abrogation of cryoablation-induced tumor regression.

“depletion of CD4+ T cells resulted in partial abrogation of the observed cryoablation-induced tumor regression (figure 1C, online supplemental figure S1)”

IFN-β production in the TME of tumor-bearing STING-/- mice was similarly enhanced 3 days following cryoablation as in wild-type mice.

“IFN-β production in the TME of tumor-bearing STING-/- mice were similarly enhanced 3 days following cryoablation as in wild-type mice (figure 3A, online supplemental figure S4F).”

• AssumptionRests on an assumption: STING-/- mice lack functional host STING signaling.

Cryoablation of established RMS tumor in STING-/- mice resulted in a significant partial reduction in tumor size over the subsequent 2 weeks, although not as robust as in wild-type mice.

“cryoablation of established RMS tumor in STING-/- mice resulted in a significant partial reduction in tumor size over the subsequent 2 weeks, although there was not as robust of a response as seen in wild-type mice (figure 3E).”

• AssumptionRests on an assumption: STING-/- mice lack functional host STING signaling.

A significant inhibition in RMS 76-9 lung metastases was observed in STING-/- mice bearing RMS 76-9STINGwt tumor.

“We also observed a significant inhibition in RMS 76-9 lung metastases in the same experimental group (figure 5B,C).”

• AssumptionRests on an assumption: STING-/- mice lack functional host STING signaling.

Cryoablation of RMS 76-9STINGwt tumor in wild-type mice produced robust systemic immunity to suppress distant lung metastasis.

“but also produced a robust systemic immunity to suppress distant lung metastasis (figure 4B,C).”

Cryoablation of RMS 76-9 tumors significantly increases Ifn-β mRNA transcript and protein secretion in tumor-infiltrating DCs.

“Tumors treated with cryoablation induced a significant increase in Ifn-β mRNA transcript (figure 3B) and protein secretion (figure 3C) in tumor-infiltrating DCs.”

A significant tumor growth inhibition was observed when cryoablation was carried out in STING-/- mice bearing RMS 76-9STINGwt tumor.

“a significant tumor growth inhibition was again observed when cryoablation was carried out in STING-/- mice bearing RMS 76-9STINGwt tumor (figure 5A).”

• AssumptionRests on an assumption: STING-/- mice lack functional host STING signaling.

Cell lysates from cryoablated RMS 76-9 cells stimulated maturation of CD11c+ DCs, as evidenced by a significant increase in the frequency of co-cultured BMDCs expressing CD86, CD40 and MHC II compared with BMDCs co-cultured with non-cryoablated RMS 76-9 cells.

“Cell lysates from cryoablated RMS 76-9 cells were able to stimulate the maturation of CD11c+ DCs, as evidenced by a significant increase in the frequency of co-cultured BMDCs expressing CD86, CD40 and MHC II, as compared with BMDCs co-cultured with non-cryoablated RMS 76-9 cells (figure 2A).”

The basal level of various lymphocyte compartments was significantly reduced or trending toward a reduction in C57BL/6 mice bearing RMS 76-9STING-/- tumors compared with RMS 76-9STINGwt.

“the basal level of various lymphocyte compartments was significantly reduced or trending toward a reduction in C57BL/6 mice bearing RMS 76-9STING-/- tumors as compared with RMS 76-9STINGwt.”

Other forms of RMS 76-9 cell death, such as radiation and chemotherapy, resulted in similar levels of MHC II and CD86 expression on co-cultured BMDCs as compared with non-cryoablated RMS76-9 cells.

“Other forms of RMS 76-9 cell death, such as those induced by radiation and chemotherapy, resulted in similar levels of MHC II and CD86 expression on co-cultured BMDCs as compared with non-cryoablated RMS76-9 cells (online supplemental figure S2).”

Co-culture of in vitro cryo-treated RMS 76-9 cells with BMDCs from STING-/- mice resulted in complete absence of Ifn-β transcription compared with BMDCs from wild-type mice.

“We co-cultured in vitro cryo-treated RMS 76-9 cells with BMDCs derived from STING-/- mice, and observed a complete absence of Ifn-β transcription as compared with BMDCs from wild-type mice (figure 3D)”

The differences among Foxp3+ CD4+ cells, NK cells, CD11c+ myeloid cells and CD11b+ myeloid cells were also lost in the context of STING-/- mice.

“The differences among Foxp3+ CD4+ cells, natural killer (NK) cells, CD11c+ myeloid cells and CD11b+ myeloid cells were also lost in the context of STING-/- mice.”

• AssumptionRests on an assumption: STING-/- mice lack functional host STING signaling.

RMS 76-9STINGwt primary tumor exhibited a robust response to cryoablation despite the absence of host STING signaling.

“RMS 76-9STINGwt primary tumor again exhibited a robust response to cryoablation despite the absence of host STING signaling (figure 8E, yellow)”

• AssumptionRests on an assumption: STING-/- mice lack functional host STING signaling.

No significant differences in the level of cell death following cryoablation in vivo within the non-necrotic tumor zone 3 days following cryoablation, as assessed by Caspase-3+ and TUNEL+ tumor cells.

“we assessed the level of Caspase-3+ and TUNEL+ tumor cells in vivo within the non-necrotic tumor zone 3 days following cryoablation, and found no significant differences in the level of cell death following cryoablation (online supplemental figure S9D,E).”

Comparing lung metastatic lesions following cryoablation, the percentage of RMS 76-9 STING-/- E2-Crimson cells in the lung was proportionally higher than that of RMS 76-9 STINGwt mNeonGreen cells.

“Comparing lung metastatic lesions in the lungs following cryoablation, the percentage of RMS 76-9 STING-/- E2-Crimson cells in the lung was proportionally higher than that of RMS 76-9 STINGwt mNeonGreen cells (online supplemental figure S10F)”

Cryoablation of primary RMS 76-9STINGwt tumor led to a significant reduction of the advanced, untreated secondary tumor.

“cryoablation of primary RMS 76-9STINGwt tumor led to a significant reduction of the advanced, untreated secondary tumor (figure 8C, yellow).”

A slightly enhanced susceptibility of STING-/- cells to cell death both in vitro and in vivo was observed.

“we also observed a slightly enhanced susceptibility of these cells to cell death both in vitro and in vivo.”

This abscopal effect on secondary RMS 76-9STINGwt was lost on cryoablation of RMS 76-9STING-/- primary tumor.

“this abscopal effect on secondary RMS 76-9STINGwt was lost on cryoablation of RMS 76-9STING-/- primary tumor (figure 8C, black).”

The absence of STING in either the tumor cells or the host alone partially suppressed tumor growth.

“the absence of STING in either the tumor cells (figure 9A, black) or the host (figure 9B, yellow) alone partially suppressed tumor growth”

CD8+ T cells were absolutely essential for the in vivo antitumor efficacy.

“CD8+ T cells were absolutely essential for the in vivo antitumor efficacy.”

No significant differences in necroptosis resulting from cryoablation between the two cell lines as assessed by phosphorylation of MLKL.

“we did not observe any significant differences in necroptosis resulting from cryoablation between the two cell lines as assessed by phosphorylation of MLKL (online supplemental figure S9A).”

A slight baseline proliferative advantage of STING-/- tumors was observed.

“Although we did observe a slight baseline proliferative advantage of STING-/- tumors, we also observed a slightly enhanced susceptibility of these cells to cell death both in vitro and in vivo.”

Genetic deletion of Tmem173 within RMS 76-9 completely abrogated cryoablation-induced IFN-β upregulation, indicating a STING dependency of this process within RMS 76-9.

“Genetic deletion of Tmem173 within RMS 76-9 completely abrogated cryoablation-induced IFN-β upregulation, indicating a STING dependency of this process within RMS 76-9.”

No significant difference in the growth of STINGwt and STING-/- tumors in vivo at baseline was observed.

“We did not observe any significant difference in the growth of STINGwt and STING-/- tumors in vivo at baseline.”

Cryoablation had no effect on the proportion of RMS 76-9 STING-/- E2-Crimson tumor cell metastases in the lung tissue compared with non-cryoablated controls.

“while cryoablation had no effect on the proportion of RMS 76-9 STING-/- E2-Crimson tumor cell metastases in the lung tissue compared with non-cryoablated controls (online supplemental figure S10E).”

Both tumors showed similar growth suppression when subjected to various cryoablation treatment regimens in vitro.

“both tumors showed similar growth suppression when subjected to various cryoablation treatment regimens in vitro (online supplemental figure S8C).”

No significant differences in cleaved Caspase-3 and Gasdermin D activation following cryoablation in vitro by western blot analysis.

“we failed to observe any significant differences in cleaved Caspase-3 and Gasdermin D activation following cryoablation in vitro by western blot analysis (data not shown).”

An abscopal effect was observed in the co-injection model.

“This conclusion was further supported by the observed abscopal effect in the co-injection model.”

In STING-/- mice, the tumor reduction efficacy of cryoablation in the primary tumor was completely lost in the context of RMS 76-9STING-/- tumor.

“The tumor reduction efficacy of cryoablation in the primary tumor was completely lost in the context of RMS 76-9STING-/- tumor (figure 8E, black).”

• AssumptionRests on an assumption: STING-/- mice lack functional host STING signaling.

Cryoablation of primary RMS 76-9STINGwt tumor led to a significant reduction of the advanced, untreated secondary tumor in the complete absence of host STING signaling.

“cryoablation of primary RMS 76-9STINGwt tumor led to a significant reduction of the advanced, untreated secondary tumor (figure 8F, yellow) in the complete absence of host STING signaling.”

• AssumptionRests on an assumption: STING-/- mice lack functional host STING signaling.

Analysis of different phases of apoptotic cell death over a 3-day time span revealed that RMS 76-9STINGwt have a higher percentage of live tumor cells, while RMS 76-9STING-/- exhibited more early and late apoptotic markers across time and cryoablation regimens.

“analysis of different phases of apoptotic cell death over a 3-day time span revealed that RMS 76-9STINGwt have a higher percentage of live tumor cells, while RMS 76-9STING-/- exhibited more early and late apoptotic markers across time and cryoablation regimens (online supplemental figure S8D-F).”

This differential STING expression between tumor and mononuclear cells was lost in the higher grade and more advanced stage tumors.

“this differential STING expression between tumor and mononuclear cells was lost in the higher grade and more advanced stage tumors (online supplemental figure S11F)”

Tmem173 and Tbk1 transcript expression in RMS were lower in metastatic and relapsed patients when compared with primary tumor at diagnosis.

“Tmem173 and Tbk1 transcript expression in RMS were lower in metastatic and relapsed patients when compared with primary tumor at diagnosis (online supplemental figure S11B)”

Mononuclear cells expressed higher levels of STING in the TME than the tumor.

“we found that mononuclear cells expressed higher levels of STING in the TME than the tumor (online supplemental figure S11E).”

Increased production of IFN-β by DCs after tumor cryoablation in vitro and in vivo.

“observed increased production of IFN-β by DCs after tumor cryoablation in vitro and in vivo.”

CD4+ T cells partially contributed to immune-mediated tumor regression following cryoablation.

“While CD4+ T cells partially contributed to this immune-mediated tumor regression following cryoablation, CD8+ T cells were absolutely essential for the in vivo antitumor efficacy.”

IFN-β production by APCs in our model is STING-dependent.

“we used BMDCs from STING-/- mice to demonstrate the STING dependency of IFN-β production by APCs in our model.”

Increased IFN-β in the TME following tumor cryoablation.

“Here, we showed increased IFN-β in the TME following tumor cryoablation, and observed increased production of IFN-β by DCs after tumor cryoablation in vitro and in vivo.”

DMXAA administration effectively reduced RMS 76-9STINGwt growth in C57BL/6 mice.

“while DMXAA administration effectively reduced RMS 76-9STINGwt growth in C57BL/6 mice (figure 9A, yellow)”

This abscopal effect on secondary RMS 76-9STINGwt was completely abrogated on cryoablation of RMS 76-9STING-/- primary tumor.

“this abscopal effect on secondary RMS 76-9STINGwt was completely abrogated on cryoablation of RMS 76-9STING-/- primary tumor (figure 8F, black).”

Successful cryoablation therapies against advanced tumor growth require the adaptive immune response.

“Using the RMS 76-9 sarcoma tumor model, we demonstrated that successful cryoablation therapies against advanced tumor growth requires the adaptive immune response, as cryoablation failed to control primary tumor growth in immune-deficient mice resulting from rapid local recurrence and metastasis following cryoablation.”

A complete absence of STING in both tumor cells and the host completely abolished in vivo antitumor efficacy of STING agonism.

“while a complete absence of STING in both tumor cells and the host completely abolished in vivo antitumor efficacy of STING agonism (figure 9B, black).”

A significant decrease in the percentage of RMS 76-9 STINGwt mNeonGreen cell subpopulation was observed in the lungs of mice treated with cryoablation compared with sham-ablated control mouse lung.

“A significant decrease in the percentage of RMS 76–9 STINGwt mNeonGreen cell subpopulation was observed in the lungs of mice treated with cryoablation as compared with sham-ablated control mouse lung (online supplemental figure S10D)”

Patient-derived data showed a wide variation in the expression level of STING pathway components in pediatric and adolescent and young adult (AYA) cancers.

“These patient-derived data showed a wide variation in the expression level of STING pathway components in pediatric and adolescent and young adult (AYA) cancers.”

Partial blunting of tumor growth was consistently observed in the complete absence of host STING signaling.

“we consistently observed a partial blunting of tumor growth in the complete absence of host STING signaling.”

IFN-β levels remained at baseline in cryoablated STING-/- RMS TME even in wild-type mice.

“whereas INF-β levels remained at baseline in cryoablated STING-/- RMS TME even in wild-type mice.”

Elevated IFN-β levels following cryoablation of RMS STINGwt irrespective of host STING status.

“Importantly, when analyzing chemokine and cytokine productions within TME, we found elevated IFN-β levels following cryoablation of RMS STINGwt irrespective of host STING status”

Increases in several chemokines and cytokines (eg, CXCL9, CXCL10, CCL6, CCL8, CCL9/10, IL-16, and CXCL2) in the RMS STINGwt TME following cryoablation even within the context of a wild-type mouse.

“Indeed, chemokine and cytokine arrays of various TME revealed increases in several chemokines and cytokines (eg, CXCL9, CXCL10, CCL6, CCL8, CCL9/10, IL-16, and CXCL2) in the RMS STINGwt TME following cryoablation even within the context of a wild-type mouse.”

Lung metastasis following cryoablation is critically dependent on a functioning tumor cell-intrinsic STING signaling pathway.

“we show that local tumor control, lung metastasis, and the abscopal effect on distant tumor are all critically dependent on a functioning tumor cell-intrinsic STING signaling pathway, which induces inflammatory chemokine and cytokine responses in the cryoablated TME.”

Local tumor control following cryoablation is critically dependent on a functioning tumor cell-intrinsic STING signaling pathway.

“we show that local tumor control, lung metastasis, and the abscopal effect on distant tumor are all critically dependent on a functioning tumor cell-intrinsic STING signaling pathway, which induces inflammatory chemokine and cytokine responses in the cryoablated TME.”

Tumor cell-intrinsic STING pathway is a critical component underlying the effectiveness of cryoablation.

“Tumor cell-intrinsic STING pathway is a critical component underlying the effectiveness of cryoablation”

Cryoablation efficacy is dependent on adaptive immunity.

“The results show that cryoablation efficacy is dependent on both adaptive immunity and the STING signaling pathway.”

• Also stated in: § Cryoablation promotes RMS regression through adaptive immunity · hedged

  “indicating that the adaptive T-cell response was required for the observed therapeutic efficacy of cryoablation in vivo (figure 1B).”

• Also stated in: § Cryoablation promotes RMS regression through adaptive immunity · boosted

  “Taken together, these data underscore the importance of host adaptive T-cell immunity in mediating a seemingly curative antitumor effect using a cryoablation procedure that proved to be only sublethal against the tumor without a functional adaptive T-cell immunity in this model.”

The reliance on tumor cell-intrinsic STING extends beyond cryoablation to include intratumoral STING agonist therapy.

“This reliance extends beyond cryoablation to include intratumoral STING agonist therapy.”

The abscopal effect on distant tumor following cryoablation is critically dependent on a functioning tumor cell-intrinsic STING signaling pathway.

“we show that local tumor control, lung metastasis, and the abscopal effect on distant tumor are all critically dependent on a functioning tumor cell-intrinsic STING signaling pathway, which induces inflammatory chemokine and cytokine responses in the cryoablated TME.”

Tumor cell-intrinsic STING signaling induces inflammatory chemokine and cytokine responses in the cryoablated TME.

“we show that local tumor control, lung metastasis, and the abscopal effect on distant tumor are all critically dependent on a functioning tumor cell-intrinsic STING signaling pathway, which induces inflammatory chemokine and cytokine responses in the cryoablated TME.”

Cryoablation efficacy is dependent on the STING signaling pathway.

“The results show that cryoablation efficacy is dependent on both adaptive immunity and the STING signaling pathway.”

Expression of STING-related signaling components may serve as a potential therapy response biomarker.

“suggests that expression of STING-related signaling components may serve as a potential therapy response biomarker.”

The STING signaling pathway is essential for tumor cryoablation-induced type I IFN production in DCs.

“suggesting that the STING signaling pathway is essential for tumor cryoablation-induced type I IFN production in DCs.”

Tumor cryoablation induces APC maturation toward immune stimulating and antitumor phenotypes.

“Our results suggest that tumor cryoablation induces APC maturation toward immune stimulating and antitumor phenotypes.”

This provides a cellular mechanism of partial cryoablation efficacy afforded by cryoablation of RMS 76-9STINGwt.

“thereby providing a cellular mechanism of partial cryoablation efficacy afforded by cryoablation of RMS 76-9STINGwt (figure 5A).”

• AssumptionRests on an assumption: STING-/- mice lack functional host STING signaling.

The increase in lung metastases suggests ineffective systemic immunity.

“suggesting ineffective systemic immunity.”

RMS 76-9 cells have an intact STING signaling pathway.

“we validated that there was an intact STING signaling pathway in RMS 76-9”

There is a generalized vital role of tumor cell-intrinsic STING in generating effective antitumor immunity in other ICD-inducing cancer therapies.

“suggesting a generalized vital role of tumor cell-intrinsic STING in generating effective antitumor immunity in other ICD-inducing cancer therapies.”

This reflects a decrease in overall STING expression, supported by the Tmem173 transcript expression profile of the PeCan dataset.

“reflecting a decrease in overall STING expression, which is supported by the Tmem173 transcript expression profile of the PeCan dataset.”

Cryoablation boosted tumor antigen presentation by DCs and T cell activation and recruitment through an ICD-mediated in situ vaccine effect.

“These findings support the view that cryoablation boosted tumor antigen presentation by DCs and T cell activation and recruitment through an ICD-mediated in situ vaccine effect.”

Tumor cell-intrinsic STING signaling critically impacts the abscopal effect of the non-cryoablated advanced distant tumors as induced by cryoablation of the primary tumor.

“Taken together, tumor cell-intrinsic STING signaling critically impacts the abscopal effect of the non-cryoablated advanced distant tumors as induced by cryoablation of the primary tumor.”

Intrinsic differences of cell proliferation or various forms of cell death between STINGwt and STING-/- tumors cannot explain the observed therapeutic efficacy of cryoablation.

“Our data suggest that intrinsic differences of cell proliferation or various forms of cell death between STINGwt and STING-/- tumors cannot explain our observed therapeutic efficacy of cryoablation.”

The vital contribution of tumor-intrinsic STING signaling to generate effective local immune TME extends beyond cryoablation into other therapeutic modalities including direct intratumoral injection of STING agonist, DMXAA.

“Importantly, the vital contribution of tumor-intrinsic STING signaling to generate effective local immune TME extends beyond cryoablation into other therapeutic modalities including direct intratumoral injection of the STING agonist, DMXAA”

This supports the observation in the RMS 76-9 model that tumor cell-intrinsic STING is critical for antitumor immunity.

“supporting our observation in the RMS 76-9 model that tumor cell-intrinsic STING is critical for antitumor immunity.”

Enhanced T cell survival in tumor-bearing STING-/- mice may be a contributing factor to the efficacy of cryoablation of RMS 76-9STINGwt in STING-/- mice.

“Beyond RMS 76-9-intrinsic STING signaling directly contributing to the clinical efficacy of cryoablation observed in STING-/- mice, a contributing factor may be the enhanced T cell survival in tumor-bearing STING-/- mice”

Our current data argue for a critical role of functional tumor cell-intrinsic STING signaling as a major determinant in cryoablation-induced antitumor immunity activation and clinical responses.

“Remarkably, our current data argue for a heretofore unappreciated but critical role of functional tumor cell-intrinsic STING signaling as a major determinant in cryoablation-induced antitumor immunity activation and clinical responses”

A myeloid cell-mediated mechanism contributes to the therapeutic effects of cryoablation.

“Although these findings support the involvement of a myeloid cell-mediated mechanism that contributes to the therapeutic effects of cryoablation”

A functional tumor STING signaling pathway serves as the dominant determinant of therapy response.

“while a functional tumor STING signaling pathway serves as the dominant determinant of therapy response.”

We provided preclinical proof of concept that tumor-intrinsic STING signaling contributes critically to cryoablation-mediated tumor suppression in vivo in at least three different tumor models, although with varying degree of efficacy.

“We provided preclinical proof of concept that tumor-intrinsic STING signaling contributes critically to cryoablation-mediated tumor suppression in vivo in at least three different tumor models, although with varying degree of efficacy.”

Tumor cell-intrinsic STING-related signaling expression is a biomarker of potential therapy responses in cryoablation.

“Our results highlight the tumor cell-intrinsic STING-related signaling expression as a biomarker of potential therapy responses in cryoablation”

The tumor cell-intrinsic STING pathway is a critical contributor to both local response and the abscopal effect in cryoablation and local STING agonist therapy.

“In conclusion, we identified the tumor cell-intrinsic STING pathway as a critical contributor to both local response and the abscopal effect in cryoablation and local STING agonist therapy.”