【Tomorrow 10-11 o'clock】

【739/740 released tomorrow at noon】

Research background

Despite progress in surgical, radiotherapy and molecularly targeted therapy, the mortality rate of non-small cell lung cancer is still high. Icis targeting the programmed cell death protein ligand 1 (pd-l1) programmed cell death protein 1 (pd-1) axis promotes tumor cell killing by activating cytotoxic T cell activity. Although these inhibitors have been approved for treatment of advanced NSCLC, most patients have little clinical benefit due to the presence of multiple immunosuppressive barriers in the tumor microenvironment (tME). Therefore, it is urgent to find immunomodulatory agents that can overcome these barriers and improve the efficacy of Icis.RT has potential immunomodulatory properties because it can trigger immunogenic cell death and activate antigen-presenting cells, causing tumor-specific T cell cross-activation, which can not only limit the growth of primary tumors, but also produce targeted at distant (non-internal).

Systemic effects of metastases. Unfortunately, the optimal RT dose and underlying molecular mechanism depend on the type of cancer, and little is known for NSCLC, including sequencing and dose/segment protocols that induce optimal immunomodulation. Emerging evidence supports the possibility of combination therapy of ICIS and RT in various tumor types including NSCLC. A phase i trial showed that NSCLC patients who had previously received radiation had a longer progression-free survival in the absence of ICIS than patients who had not received RT. However, due to the lack of identified molecular mechanisms in previous trials, current clinical trials tested this combination as directed by empirical choices, which may not be optimal. Here, we provide mechanical insights that may accelerate the development of RT and ICIS combination therapy in NSCLC.

?Technical route?

picture

?Research results?

Specific doses of RT enhance the efficacy of pd-1 blockers

picture

In ongoing studies of human and murine tumors, different doses of low segmentation RT have been reported and the key importance of dose and segmentation has been proposed. Therefore, we first optimized the HKP1 tumor-bearing lung RT protocol under microcomputed tomography (μct)-guided, which could improve the therapeutic effect of PD-1 inhibition. We tested an RT protocol previously reported with immunomodulatory activity in breast cancer models: 8gy for 3 consecutive days (8gy×3), and two lower dose regimens, 4gy×3,

and 0.5gy×3 (Figure 1a). It is worth noting that 4gy×3 (4gy-rt) combined with pd-1 antibody can significantly control tumors (Figure 1b,c) and improve survival (Figure 1d). In addition, 40% of mice survived tumor-free, while the tumor-free survival rate in cohort mice treated with pd-1 antibody was 10%. Compared with inhibiting pd-1 alone, other doses of 0.5gy×3 and 8gy×3 combined with pd-1 inhibition did not significantly control tumors or improve survival (Figure 1e,f).

Obtaining durable T-cell activity through combination therapy

picture

To elucidate the different effects of different radiotherapy doses, we detected T cell infiltration and cytokine production 1d after the last radiotherapy (day 4 after the start of radiotherapy). Among all the cohorts examined, 4gy-rt showed the highest number of CD8T cells invaded on tumor islets (Figure 2a and extended data Figure 1a), and the highest number of interferon-γ/tumour necrosis factor-α/cd4t cells and gzmb/cd8t cells in the HKP1 lungs (Figure 2b,c and extended data Figure 1b). These 4gy-rt-induced T cell activity is consistent with its inhibition of PD-1 synergy. More and more T cells are recruited to the HKP1 lungs and gradually obtain dysfunctional phenotypes, manifested as reduced effector cytokine expression in CD4t cells, such as interferon-γ and tumor necrosis factor-α (Figure 2a,b). To determine 4gy

Whether RT enhances the function of the original T cells, we treated HKP1 mice with the S1P receptor inhibitor Fty720, which prevented activated T cells from flowing out of the lymph nodes (Extended Data Figure 2c). As expected, Fty720 treatment restricted the effect of 4gy-rt to the original T cells in the lungs by reducing circulating T cells (Extended Data Figure 2d). Compared with the control group, Fty720 canceled the ability of 4gy-rt to increase interferon-γ/tumor necrosis factor-α/cd4t cells and gzmb/cd8t cells (Extended Data Figure 2e). Therefore, in the mice treated with Fty720, the effect of 4gy-rt and anti-PD-1 was abolished (Extended Data Figure 2f). These findings suggest that 4gy-rt does not reactivate the original dysfunctional T cells in TME.

4gy-rt therapy activates lung resident rod cells in TME

picture

To explore the mechanism by which 4gy-RT induces anti-tumor immune responses, we examined RT-induced activation of dendritic cells (DC). 4gy-RT treatment neither caused tumor cell death (Extended Data Fig. 4a) nor increased CC cross-presentation of Cd103 in bronchial lymph nodes (Extended Data Fig. 4b). Next, we performed RNA-seq analysis of HKP1 lungs treated with RT (0gy, 4gy or 8gy×3) combined with IGG or PD-1 antibody. Comparison of differentially expressed genes in the 4gy-RT group, 0gy (simulated) group and 8gy-RT (invalid dose) group before and after anti-PD-1 treatment (p<0.0    1, doubling ≥2 and least squares mean ≥5). By overlapping the 4gy-rt upregulated genes in the IGG cohort with the anti-PD-1 cohort, we identified 144 genes specific to upregulation of this effective radiation dose (Figure 3a and extended data Figure 5a). To determine the cell origin of these 4 gy-rt characteristic genes, we performed single-cell RNA-seq (scRNA-seq) analysis of HKP1 tumor-bearing lungs receiving 0gy or 4gy-rt. By projecting 144 signature genes onto the T-distribution random neighbor embedding (t-sne) plot, we found that these genes were significantly upregulated in the airway epithelial/rod-like cluster after 4gy-rt (Figure 3b).

Rod cell secretomes are related to the efficacy of combined treatment

picture

Regardless of the depletion method, the immune response of the lungs carrying hkp1 to 4gy-rt was significantly weakened, manifested as significantly weakening of T cells infiltration into tumor pancreatic islets, and the production of effector cytokines was reduced (interferon-γ/tumor necrosis factor-α in cd4t cells and gzmb in cd8t cells) (Figure 4c and extended data Figure 6d). It is worth noting that in hkp1 tumor-bearing mice, rod-shaped cell defects abolished the efficacy of combined treatment (Figure 4d).

picture

Further characterization of rod-shaped cells by electron microscopy showed that the number of secreted vesicles in rod-shaped cells isolated from 4gy-rt-t-treated lungs compared with the control group (Extended Data Figure 8a, b). We speculated that the secretion of rod-shaped cells after radiotherapy may play a role in improving ICIS efficacy. To solve this problem, we used scgb1a1-creertm/snap23flox/flox transgenic mice (referred to as scgb1a1creer/snap23fl/fl) to prevent rod-shaped cells from entering the lung TME. The synaptic-associated protein 23 (snap23) is a key component of the Snare complex and mediates fines. Intracellular vesicles fuse onto the membrane to regulate exocytosis. Tamoxifen treatment of scgb1a1cre/snap23fl/fl mice allowed conditional rod-shaped cells to specifically deletion of the snap23 gene (Figure 5a) without altering the integrity of the bronchiole (Extended Data Figure 8c). Analysis of bronchial alveolar lavage fluid (balf) confirmed that 4gy-rt-treated scgb1a1cre/snap23fl/10 was significantly reduced compared to wild-type controls (Figure 5b). Consistent with rod-shaped cell ablation, snap23 deficiency inhibited the infiltration and activation of T cells in the HKP1 lungs in the 4gy-rt response (Figure 5c and extended Data Figure 8d).

Rod-shaped cells reduce tumor-promoting inflammation

picture

The lungs of globular cell-deficient (dt-treated) mice showed an increase in myeloid/lymphocyte ratio (Mye/lym, 0.96 and 1.34 for control and dt-treated mice, respectively, Figure 6a). In addition, myeloid cells in rod-deficient lungs exhibit increased expression of inflammatory mediators such as il1b, ptgs2 and tnf (Figure 6b), which have been reported to inhibit adaptive anti-tumor immunity. We speculated that these rod-like cell-mediated TME changes may affect the phenotype of T-cell effectors. Using graph-based classification, T-cells were divided into 9 clusters (Figure 6c), and the functional status of each cluster was determined according to their characteristic genes (Figure 6c and Supplementary Table 2). We observed the effect

The T cell cluster (C5) should show increased expression of cytokines, effector molecules (interferon-γ and tumor necrosis factor) and T cell activation markers (PDCD1 and CTLA4), the proliferating T cell cluster (C9) shows granzyme and Ki67 expression, and the regulatory T cell cluster (C8) shows Foxp3 and IL2RA expression (Figure 6c). It is worth noting that compared with the control group (2.63 and 1.96, respectively), derodrotor cells (DT treatment) reduced the ratio of effector, proliferating T cells and Treg cells ((c5c9)/c8). In summary, these scRNA-seq results show that the presence of RT-activated rod cells contributes to adaptive anti-tumor immunity.

Elisa detection of the balf sample showed that 4gy-rt significantly reduced the levels of il-1β, pge_2 and tnf-α in HKP1   TME compared with the 0gy control group (Figure 6D), but in the absence of rod-shaped cells (DT-treated SGB1a1cre/idtr) or their secretomes (SGB1a1cre/snap23fl/fl) (Figure 6D). To demonstrate the clinical relevance of RT-induced rod-shaped cell secretomes, we failed to reduce these inflammatory factors in the phase II neoadjuvant clinical trial (NCT0)

Plasma levels in NSCLC patients treated with RT combined with ICIS (anti-PD-L1) were evaluated in 2904954)54. Plasma was collected 1d before and 1d after the first stereotactic systemic radiation therapy (Sbrt) and 1d after the last Sbrt. Comparison of plasma concentrations of 10 (before and after radiotherapy) showed that 10 levels were elevated (5 out of 8 people) while unresponsive patients (0 out of 9 people, p=0.0301) (Figure 6e), indicating that rod cell activation is related to the pathological response of combined treatment. In summary, these findings indicate that RT-activated rod cell cells inhibit pre-tumor inflammation in HKP1 lungs through their secretions, thereby enhancing adaptive anti-tumor immunity.

Rod-shaped cytokines improve the efficacy of pd-1 receptor blockers

picture

These in vitro research results prompted us to study whether rod-shaped cocktails can reflect the effect of 4gy-rt in improving PD-1 blockade in vivo. We injected rod-shaped cocktails into HKP1 mice intranasally (20   ng per protein per mouse) for 3 days (days 11-13), and combined with PD-1 antibody (Figure 8a). It is worth noting that compared with simulated controls, rod-shaped cocktail treatment with PD-1 antibody can significantly control tumors and improve the survival rate of HKP1 mice (Figure 8b,c). Similar to 4gy-rt, bronchial alveolar lavage fluid analysis showed that

, Compared with the control group, the mice in the rod-shaped cocktail treatment group had lower levels of tumor necrosis factor-α, prostaglandin e_2 and il-1β (Figure 8D). Finally, to determine whether combined treatment of rod-shaped cocktail and pd-1 antibody can produce lasting anti-tumor immunity, we conducted tumor re-attack experiments. In fact, compared with the control group, the surviving mice cleared tumor cells from the lungs within 3-5 days (Extended Data Figure 10E). In summary, these findings suggest that rod-shaped cell secreted proteins improve the efficacy of pd-1 blockade by inhibiting immunosuppressed mdscs and enhancing T-cell function.

?Research summary?

In the nsclc model, a specific dose of RT was identified that when used in combination with ICIS, which can regress tumors and increase survival.

The immunomodulatory effect of RT is attributed to activated lung residency of SGB1A1 rod-shaped cells. It is worth noting that mice with rod-shaped cell-specific synaptic-associated protein 23 knockout failed to benefit from the combination therapy, suggesting that rod-shaped cell secretions play a key role.

We identified eight rod-shaped cell secreted proteins that can inhibit immunosuppressed myeloid cells, reduce pre-tumor inflammation, and enhance anti-tumor immunity. Notably, the 10 levels are elevated.

?discuss?

Our observations are consistent with the mainstream view that RT combined with ICIS can elicit anti-tumor responses. It is worth noting that our study provides a mechanism to enhance the therapeutic effect of ICIS on non-small cell lung cancer by induced sluggish sterilization of SCGB1A1 rod-shaped cells through radiation. There is a growing body of evidence that the immunosuppressive microenvironment associated with pro-cancer inflammation constitutes the main obstacle to the success of cancer immunotherapy, and proposes suggestions for targeting inflammation to improve the efficacy of ICIS. With the reported SLR

The anti-inflammatory effects of α cells in allergies, asthma and copd are consistent, and we demonstrate the intriguing role of RT-activated rod-cell secreted proteins in non-small cell lung cancer, which can combat multiple immunosuppressive mediators and enhance adaptive anti-tumor immunity. The insights obtained from our study have significant potential in clinical translation. Currently, standards for switching radiation doses from animal models to humans and vice versa are lacking due to differences in radiation sensitivity and target size in specific species. However, I

Our study shows that future clinical trials of human non-small cell lung cancer should consider optimizing a radiation therapy dose that can effectively mediate the activation of resident rod cells in the lungs. Currently, there is a lack of pharmacological or genetic approaches to activate rod cells without compromising the integrity of the lung epithelial. It demonstrates that the administration of rod cell cocktails enhances the efficacy of icis, with great potential in developing these factors as a treatment for non-small cell lung cancer. Further research requires identification of secretions that can produce strong therapeutic effects.

Optimal composition of factors. Notably, SBT increases the level of neoadjuvant SBT10, a finding that suggests that RT-induced rod-cell secretion factors in plasma may be likely to serve as non-invasive biomarkers that respond/unresponsive to SBT/ICIS treatment. In summary, insights from our study have the potential to provide guidance for future clinical trials to maximize the effectiveness of the combination of radiation and immunotherapy to improve pathological response rates and improve survival in patients with non-small cell lung cancer.
Chapter completed!