Combining a nanoparticle-mediated immunoradiotherapy with dual blockade of LAG3 and TIGIT improves the treatment efficacy in anti-PD1 resistant lung cancer | Journal of Nanobiotechnology

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Dual blockade of LAG3 and TIGIT improves treatment outcome of NBTXR3-mediated immunoradiotherapy

To address the upregulation of LAG3 and TIGIT induced by the treatment of NBTXR3 + XRT + αPD1, we established a two-tumor model with 344SQR αPD1-resistant lung cancer in mice, which were subsequently treated with various combinations of radiation (XRT), XRT enhanced with NBTXR3, αPD1, αLAG3, and αTIGIT (Fig. 1A). Consistent with our previously published results [20], irradiation of tumors injected with NBTXR3 and treated with αPD1 produced superior control of tumor growth and longer animal survival time than XRT + αPD1 without nanoparticle injection (Fig. 1B). The combination of triple checkpoint blockade (αPD1 + αLAG3 + αTIGIT, hereafter abbreviated PLT) in the absence of any radiotherapy (XRT alone or NBTXR3 + XRT) did not achieve significant control of either the primary or the secondary tumors. In addition, the co-blockade of LAG3 and TIGIT did not enhance treatment outcomes of XRT + αPD1 without NBTXR3. However, adding NBTXR3 + XRT + PLT led to significantly slower growth of both the primary and the secondary tumors as well as extended survival. The median survival times of each group, in days, were as follows: control (16), XRT + αPD1 (21), PLT (17), XRT + PLT (24), NBTXR3 + XRT + αPD1 (25), and NBTXR3 + XRT + PLT (35) (Fig. 1B, C). NBTXR3 + XRT + PLT markedly slowed the tumor growth in most of the treated mice, and in 25% (2 out of 8) of the mice that received NBTXR3 + XRT + PLT, the tumors were completely eradicated (Additional file 1: Fig. S2). In contrast, no mice from any of the other treatment groups survived the entire assay. Although NBTXR3 + XRT + αPD1 was effective in delaying the growth of both the primary and secondary tumors in most of the mice, it was ultimately unable to stop tumor growth in any of them.

Having established the superiority of NBTXR3 + XRT + PLT, we next sought to evaluate the benefit of adding either αLAG3 or αTIGIT individually to NBTXR3 + XRT + αPD1. Either αTIGIT or αLAG3 was able to significantly improve control of both the primary and the secondary tumors, and no significantly different treatment efficacy was observed between NBTXR3 + XRT + αPD1 + αLAG3 and NBTXR3 + XRT + αPD1 + αTIGIT in terms of tumor growth or survival (Fig. 1C). However, neither of these two combination therapies achieved remission of the tumors. In contrast, in this particular survival assay, treatment with NBTXR3 + XRT + PLT resulted in 3 of the 8 mice (37.5%) being entirely cured from their tumors (Fig. 1C).

Previously, we observed that improved control of primary and secondary tumors was accompanied by fewer lung metastases [20]. To confirm this result in our present study, we counted the number of metastatic lesions in the lungs of our mice on day 21. In keeping with our prior observations, lung metastasis corresponded sharply with control of the primary and secondary tumors (Additional file 1: Fig. S3). Every treatment group paired NBTXR3 with any combination of CPIs significantly reduced the number of spontaneous lung metastases compared to the control. The addition of either αLAG3 or αTIGIT to NBTXR3 + XRT + αPD1 resulted in significantly fewer lung metastases, and the addition of both in concert achieved the lowest numbers of metastases of any treatment group.

Lastly, we monitored the body weight of the mice implanted with 344SQR tumors followed by treatment with NBTXR3 + XRT + PLT and those untreated naïve mice, no significant difference in body weight was observed between the two groups (Additional file 1: Fig. S4).

The treatment efficacy of NBTXR3 + XRT + CPIs is heavily dependent on immune cells

The abscopal effect is thought to be mediated by the immune response [22]. To elucidate if the treatment benefits we observed in our NBTXR3 + XRT + PLT treatment group were indeed immune-mediated and, if so, what populations of immune cells were involved in the antitumor activity, we depleted CD4+ T cells, CD8+ T cells, and NK cells with antibodies from mice in this treatment group. The depletion of CD4+ T cells, CD8+ T cells, and NK cells all detrimentally impacted the treatment efficacy of the NBTXR3 + XRT + PLT therapy, but at different levels (Fig. 2). CD4+ T cell depletion completely ablated the tumor control efficacy of the NBTXR3 + XRT + PLT therapy, resulting in tumor growth and survival curves virtually indistinguishable from those of untreated controls (Fig. 2A–C). Depletion of CD8+ T cells impaired but did not entirely ablate control of primary tumor growth and overall survival in the NBTXR3 + XRT + PLT group; like with CD4+ depletion, however, secondary tumor growth was completely unrestrained. NK cell depletion had the least impact, with primary tumor growth curves only slightly inferior to treated mice who were not immunodepleted; the difference in secondary tumor volume was more substantial, but still the least affected of the immuno-depleted treatment groups. The survival time of NK-depleted mice was slightly improved over those of CD8-depleted. In any case, all immunodepletion interventions impaired the treatment efficacy of NBTXR3 + XRT + PLT. Two out of the 5 mice from the non-immunodepleted group survived the entire experiment; none of the others did. Our results demonstrate that CD4+ T cells, CD8+ T cells, and NK cells are all essential to the efficacy of the NBTXR3 + XRT + PLT treatment, with CD4+ T cells being the most indispensable and NK cells the least.

Fig. 2
figure 2

The treatment efficacy of NBTXR3 + XRT + PLT (Combo) heavily depends on immune cells. Mice (n = 5) were treated by the combination therapy of NBTXR3 + XRT + PLT, as described in Fig. 1. In addition, αCD4 (500 µg), αCD8 (500 µg), and αAsialo GM1 (30 µL) antibodies were given via intraperitoneal injection on days 5, 7, 9, and 12, and 17 to deplete CD4+ T cells, CD8+ T cells, and NK cells, respectively. Mice were euthanized when the primary or the secondary tumors reached 14 mm in any dimension. A Tumor volume of the primary tumor. B Tumor volume of the secondary tumor. C Survival rates of the mice. Tumor volumes were compared by two-way analysis of variance and were expressed as mean tumor volume ± standard error of the mean ± SEM. Mouse survival rates were analyzed with the Kaplan–Meier method. P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, not significant

Dual blockade of LAG3 and TIGIT increases proliferation of CD4+ and CD8+ T cells

By now, we had established two points: that mice treated with NBTXR3 + XRT + PLT could control tumor growth and survive in a way that XRT + PLT treated mice were unable to match, and that this ability was dependent upon T cells. Given these two points, we decided to examine what effect this treatment might have on these immune cell populations. TIGIT and LAG3 are well-known to induce T cell exhaustion. Thus, we reasoned that these two markers’ blockade might alleviate T cell exhaustion.

To evaluate this hypothesis, we analyzed the proliferation of both CD4+ and CD8+ T cells, as assessed by Ki67 expression, in mice treated with XRT + αPD1, NBTXR3 + XRT + αPD1, NBTXR3 + XRT + αPD1 + αLAG3, NBTXR3 + XRT + αPD1 + αTIGIT, and NBTXR3 + XRT + PLT. No significant differences were detected in the percentage of CD4+Ki67+/CD4+ T cells in the primary or the secondary tumors between the experimental groups (Fig. 3A, B and Additional file 1: Fig. S5A, B, and D). In contrast, proliferating CD8+ T cells increased in the primary tumors of mice treated with αTIGIT and/or αLAG3 (Fig. 3 A and Additional file 1: Fig. S5A), a significantly higher percentage of CD8+Ki67+ T cells was observed in the secondary tumors in mice treated with either αTIGIT (but not αLAG3), and a much greater percentage was observed in the secondary tumors of mice treated with NBTXR3 + XRT + PLT (Fig. 3B and Additional file 1: Fig. S5B). In the blood, flow cytometry analysis show that NBTXR3 + XRT + PLT produced a significantly higher percentage of Ki67+CD4+ T cells than all other combination therapies and a significantly higher percentage of Ki67+CD8+ T cells than the NBTXR3 + XRT + αPD1 group, but not the NBTXR3 + XRT + αPD1 + αLAG3 or NBTXR3 + XRT + αPD1 + αTIGIT group (Fig. 3C and Additional file 1: Fig. S5C). These results show that dual blockade of LAG3 and TIGIT, in concert with NBTXR3-amplified radiation therapy and PD1 blockade, promotes the proliferation of intratumoral proliferation of CD8+ T cells and the systemic proliferation of both CD4+ and CD8+ T cells. In addition, nanostring analysis of dual blockade of LAG3 and TIGIT did not significantly change the number of CD8+ T, NK, B, and Treg cells in the primary or secondary tumors. However, the mice treated with NBTXR3 + XRT + PLT exhibited more CD45+ immune cells in the secondary tumors (Additional file 1: Fig. S7).

Fig. 3
figure 3

Dual blockade of TIGIT and LAG3 promotes proliferation of CD4+ and CD8+ T cells. A Percentages of Ki67+CD4+ and Ki67+CD8+ T cells in the primary tumors. B Percentages of Ki67+CD4+ and Ki67+CD8+ T cells in the secondary tumors. C Percentages of Ki67+CD4+ and Ki67+CD8+ T cells in the blood. The mice (n = 5) were treated with various combination therapies, including XRT + αPD1, NBTXR3 + XRT + αPD1, NBTXR3 + XRT + αPD1 + αLAG3, NBTXR3 + XRT + αPD1 + αTIGIT, and NBTXR3 + XRT + PLT as indicated in Fig. 1 A and were sacrificed on day 21. The mice which were inoculated with tumors only served as control. Immune cells from primary tumors, secondary tumors, and blood were processed and stained with αCD45-APC-Cy7, αCD3-PE-Cy7, αCD4-alexa 700, αCD8-PercpCy5.5, and αKi67-alexa 647. The cells were run with a Gallios Flow Cytometer (Beckman Coulter) and analyzed with Kaluza software Version 2.1. The data were expressed as mean ± SEM and were analyzed with a two-tailed t test. P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

NBTXR3, in tandem with triple CPIs, stimulates the activation of immunological genetic programs

Having delineated the efficacy of these combinatorial treatments in our mouse model, we next sought to parse the genetic responses to each treatment. To this end, we excised primary tumors from the mice and subjected them to complete cellular dissolution, followed by RNA extraction. We then analyzed the comparative abundance of 770 different immune-related genes using the Nanostring PanCancer Immune Profiling Advanced Analysis Module. We observed significant elevations in the expression levels of genes involved in innate immunity, humoral immunity, B cell function, DC function, and antigen processing in mice that were treated with NBTXR3 + XRT + PLT when compared to the control (Fig. 4). We also observed elevations in genes involved in adaptive immunity, T cell function, and NK cell function; however, these increases were not statistically significant. Similar elevations were observed for NBTXR3 + XRT + αPD1 and αLAG3 or NBTXR3 + XRT + αPD1 and αTIGIT, though these were not significantly higher than treatment with just NBTXR3 + XRT + αPD1. Remarkably, NBTXR3 + XRT + PLT also displayed increased activities in humoral, B cell function, and antigen processing pathways compared to XRT + αPD1(Fig. 4).

Fig. 4
figure 4

Activity score of immune pathways in the irradiated tumors. Mice (n = 4) were treated with various combination therapies as described in Fig. 1 and were euthanized 11 d post last fraction of radiation. The RNA from the irradiated tumors was extracted, and the immune-related genes were measured with a nCounter PanCancer Immune Profiling Panel and a nCounter MAX Analysis System. The data were analyzed with the PanCancer Immune Profiling Advanced Analysis Module. P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant

Given the unique efficacy we observed for NBTXR3 + XRT + PLT treatment, we specifically compared the immunogenetic profile of primary tumors treated with NBTXR3 + XRT + PLT, NBTXR3 + XRT + αPD1 and αLAG3, or NBTXR3 + XRT + αPD1 and αTIGIT as compared to those treated with NBTXR3 + XRT + αPD1 alone. In this manner, we hoped to see if each iterative addition of blockers induced any additional genetic activation. When we thus analyzed the RNA data, we observed no additional upregulation of adaptive immune-related genes with any combination of blockers above that achieved by NBTXR3 + XRT + αPD1 (Fig. 5). On the contrary, there was, in fact, a marked downregulation of two immune-related genes: Irf7, which is strongly involved in antiviral immunity [30], and C1qbp, a component of the complement protein C1q-binding receptor that is strongly associated with the promotion of chemotaxis and metastasis in several cancer types [31, 32].

Fig. 5
figure 5

Log2 fold change in expression of genes involved in adaptive, innate immunity, and T cell function in the irradiated tumors. Mice (n = 4) were treated with various combination therapies as described in Fig. 1 and were euthanized 11 d post last fraction of radiation. The RNA from the irradiated tumors was extracted, and the immune-related genes were measured with a nCounter PanCancer Immune Profiling Panel and a nCounter MAX Analysis System. The data were analyzed with the PanCancer Immune Profiling Advanced Analysis Module

When we examined innate immune genes, we saw significant downregulation of even more immune-related genes, all broadly associated with activation and inflammation (Additional file 1: Fig. S8A). These included: Irf7; interferon-stimulated genes Isg15 and 20; complement factor B (Cfb); Axl, a receptor tyrosine kinase that facilitates immune evasion and metastasis in various cancers [33, 34]; dual specificity phosphatases Dusp6 and 8; and Map2k1, which is frequently dysregulated in cancer and is the target of numerous experimental inhibitors being developed [35]. While the individual genes differed somewhat, this downregulation of the inflammatory gene was observed for all three treatments (Fig. 5).

However, in NBXTR3 + PLT alone, we observed something new: a marked upregulation in several innate-immune related genes—very one of which was in involved with macrophage activation (Additional file 1: Fig. S8A). These macrophage-associated genes included: Slamf7, a super-activator of macrophages and a strong promoter of anti-tumor phagocytosis [36]; Abcg1, a macrophage membrane transporter protein that mediates cholesterol efflux, promotes macrophage migration, and restrains inflammation and apoptosis [37,38,39]; Lgals3, a cell-cell adhesion model also involved in macrophage activation [40]; cathepsin S (Ctss), a lysosomal protease involved in peptide catalysis and antigen presentation in macrophages and DCs [41, 42]; and Itgax, a granulocyte integrin which promotes macrophage activation and anti-tumor immunity [43]. The protein product of Itgax, CD11c, also serves as the classical marker for antigen-presenting DCs [44]. Taken together, the gene expression changes within our treatment groups at the primary tumor site point to the downregulation of genetic programs involved in inflammatory and antiviral-like immunity. Moreover, when PD1, LAG3, and TIGIT were inhibited in tandem with NBTXR3-enhanced radiation, there was also a robust and unambiguous elevation of genes involved in macrophage activation, enhanced trafficking, tumor phagocytosis, and antigen presentation.

We next examined changes in immune-related genes in the secondary tumor (unirradiated). Unlike the primary tumor—in which only genes in pathways associated with innate immunity, antigen processing, and B cell function showed upregulation—in the secondary tumor treated with NBTXR2 + XRT + αPD + αTIGIT or αLAG3 or αLAG3 and αTIGIT, we observed marked, statistically robust increases activity and gene upregulation in all the immune pathways, including adaptive, T cell function, B cell function, dendritic cell function, innate, NK function, etc. (Figs. 6 and 7). This upregulation was the most pronounced in the mice treated with NBTXR3 + XRT + PLT (Additional file 1: Fig. S8B). Mice treated with NBXTR3 + XRT + αPD1 + αLAG3 universally experienced a broad “smear” of gene expression fold changes above and below that of untreated controls, possibly indicating highly dynamic up- and downregulation of several genes; however, most analyzed genes were upregulated. Upregulation was present—and much sharper and less ambiguous—in mice treated with NBXTR3 + XRT + αPD1 + αTIGIT. In both of these combinations (+αLAG3 and + αTIGIT), some genes were upregulated above that of the triple therapy. However, NBTXR3 + XRT + PLT boasted the “cleanest”, sharpest signal.

Fig. 6
figure 6

Activity score of immune pathways in the unirradiated tumors. Mice (n = 4) were treated with various combination therapies as described in Fig. 1 and were euthanized 11 d post last fraction of radiation. The RNA from the unirradiated tumor was extracted, and the immune-related genes were measured with a nCounter PanCancer Immune Profiling Panel and a nCounter MAX Analysis System. The data were analyzed with the PanCancer Immune Profiling Advanced Analysis Module. P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant

Fig. 7
figure 7

Log2 fold change in expression of genes involved in the adaptive pathway, innate pathway, T cell function, and NK cell function in the unirradiated tumors. Mice (n = 4) were treated with various combination therapies as described in Fig. 1 and were euthanized 11 d post last fraction of radiation. The RNA from the unirradiated tumor was extracted, and the immune-related genes were measured with a nCounter PanCancer Immune Profiling Panel and a nCounter MAX Analysis System. The data were analyzed with the PanCancer Immune Profiling Advanced Analysis Module

Given the upregulation of both innate and adaptive pathways – including pathways involved in DC function, antigen processing, T cell function, and B cell function, we suspected that what was occurring at the secondary tumor site was the recruitment of tumor-infiltrating lymphocytes (TILs) that had been primed by macrophages and DCs from the primary site. In the exploration of this hypothesis, we closely examined the individual genes that were statistically significantly (p > 0.05) upregulated in the NBTXR3 + XRT + PLT group and manually grouped them according to function (Additional file 2: Table S1). We then plotted the aggregate of the log2 change of each individual gene from the NBTXR3 + XRT + PLT in order to obtain a sense of which immune-related pathways were being altered following treatment (Additional file 1: Fig. S8C). Using this analysis, we found that genes involved in phagocytosis, antigen processing and presentation, cell-cell adhesion, and CD4+ T cell receptor (TCR) signaling were all elevated in the NBTXR3 + XRT + PLT group compared to the NBTXR3 + XRT + αPD1 group. Also upregulated were genes involved in various activating pathways associated with the immune response: the JAK-STAT pathway, MAP kinases, IRAKs and TRAFs, and NFκB, as well as various immune-associated tyrosine kinases. The expression of genes encoding numerous anti-inflammatory cytokines was also heightened. Among them was IFNγ, produced by activated CD4+ T cells responding to antigen recognition. In short, the genetic signature within the secondary tumor bore the unmistakable mark of robust activation of adaptive immunity through antigen presentation.

Also present was the genetic signature of a vigorous innate immune response. As previously mentioned, genes governing phagocytosis—the process whereby innate immune cells, mostly macrophages, engulf target cells—were highly upregulated. So too were genes involved in reactive oxygen species (ROS) generation, typically upregulated by activated innate immune cells to further their activation and digesting their prey engulfed through phagocytosis. Several genes involved in the complement system, a central mediator of radiotherapy-induced tumor-specific immunity [45], were also upregulated. Among the most highly upregulated gene groupings were those specifically associated with macrophage identity and function. The gene for macrophage colony-stimulating factor (Csf1) and its receptor, Csf1r, were both elevated, as were the genes for natural resistance-associated macrophage protein 1 (Slc11a1), macrophage receptor with collagenous structure (Marco), and macrophage inflammatory protein 1 β (Ccl4). We, moreover, observed an even greater upregulation of the macrophage super-activator Slamf7. Altogether, our NanoString data paint a picture of strong innate and adaptive immune responses at the secondary tumor site.

NBTXR3 + XRT + PLT treatment produces long-term immunological memory

As demonstrated in the results above, antitumor immune response plays a vital role in the NBTXR3 + XRT + PLT therapy resulted tumor eradication. The most robust immune responses are marked by the development of immunological memory, in which a small remnant of antigen-specific T cells and B cells activated in the initial antigen exposure persist, primed, and ready to respond rapidly should the organism ever be challenged by the same pathogen. The abscopal effect is thought to stimulate such a response, essentially converting the primary tumor into an in situ vaccine [46].

To evaluate if the cured mice developed an antitumor memory immune response, the 5 survivor mice from the NBTXR3 + XRT + PLT group were re-injected with 5 × 104 344SQR cells on the right flank, and their tumor growth was monitored. None of these mice developed tumors (Fig. 8A). In contrast, all mice in the control group did. Twenty-eight days post tumor re-challenge, when the mice in the control group had reached the experimental endpoint, all mice were sacrificed, and their lung metastases were counted. No lung metastasis was observed in the NBTXR3 + XRT + PLT group (Fig. 8B); however, all of the mice in the control group developed various numbers of tumor nodules in their lungs.

Fig. 8
figure 8

NBTXR3 + XRT + PLT (Combo) treated mice reject tumor re-challenge. A Tumor volume and images of the tumors in the mice. B The number of lung metastases and images of the lung tumors. The five mice that survived in the NBTXR3 + XRT + PLT group in Fig. 1 were re-challenged with 5 × 104 344SQR cells on the right flank at least 60 d after the last fraction of 12 Gy. Five mice with 5 × 104 344SQR cells inoculated on the right flank served as the control. All the mice were euthanized 28 d after tumor inoculation. Lungs were harvested from the mice, and the number of lung metastases was counted. P < 0.05 was considered statistically significant. **P < 0.01, NS, not significant

We next looked directly at the levels of total, central, and effector memory T cells (Fig. 9A). We collected blood and spleens from both experimental groups, from which we isolated CD4+ and CD8+ memory T cells. The differences in memory composition and distribution were broadly similar between both T cell subsets. For both CD4+ and CD8+ cells, total (CD45+) and effector memory T cells (TEM cells; CD44+CD62L) were elevated within the blood and spleens of NBTXR3 + XRT + PLT-treated mice (though this elevation was not statistically significant for blood total memory CD8+ T cells). Central memory T cells (TCM cells; CD44+CD62L+) of both compartments were enriched in the blood – significantly so in the case of CD8+ T cells; curiously, however, TCM cells of both compartments were less abundant within the spleens of treated mice than controls (again, this was statistically significant for CD8+ T cells, not so for CD4+). Given the context of this observation (i.e., during an ongoing immune challenge), we suspect that these numbers paint the picture of a poised memory response that has been sprung, triggering a mass re-activation of TCM cells of both subsets, their differentiation into TEM cells [47], and their exodus from the spleen into the bloodstream.

Fig. 9
figure 9

NBTXR3 + XRT + PLT (Combo) induces immunological memory and upregulates immune activities in the blood. A Percentages of CD4+/CD45+ and CD8+/CD45+ T cells and memory CD4+ and CD8+ T cells in the blood and spleen. B Nanostring activity score of immune pathways and log2 fold change in expression of genes involved in the adaptive pathway, NK cell function, B cell function, and T cell function with the control as the baseline. The five mice that survived in the NBTXR3 + XRT + PLT group in Fig. 1 were re-challenged with 5 × 104 344SQR cells on the right flank at least 60 d after the last fraction of 12 Gy. Five mice with 5 × 104 344SQR cells inoculated on the right flank served as the control. All the mice were euthanized 28 d after tumor inoculation. Blood was harvested before euthanizing the mice, red blood cells were lysed, and RNA was extracted for Nanostring analysis. The populations of memory CD4+ and CD8+ T cells were analyzed by flow cytometry. P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant

Finally, we examined peripheral blood monocytes (PBMCs) isolated for gene upregulation using the same Nanostring module previously described. PBMCs from mice treated with NBTXR3 + XRT + PLT exhibited significant upregulation in every immune-related genetic program examined, and a significant downregulation of genes associated with cancer progression (Fig. 9B). This, in coordination with the lack of any tumor development and the hardy memory T cell response, indicates that mice treated with NBTXR3 + XRT + PLT were not only able to survive and clear the initial tumor challenge, but were able to develop immunological memory that inoculated them against the same tumor type.