3.1 Construction of humanized mouse model with higher metastatic potential for neoadjuvant immunotherapy
Establishing ideal preclinical mouse models that accurately simulate postoperative recurrence and metastasis is crucial for subsequent neoadjuvant immunotherapy research. However, the low metastatic potential of common NSCLC cell lines suggests that significant postoperative recurrence and metastasis are unlikely to occur. Therefore, we initially selected NSCLC cell line and conducted screening in humanized mice to isolate its highly metastatic subpopulation. As previous studies have demonstrated that elevated PD-L1 expression in NSCLC is generally indicative of improved responses to immunotherapy [24]. We firstly detected PD-L1 expression in several human NSCLC cell lines. Among the cell lines tested, H460 and PC-9 cells displayed the highest levels of PD-L1. However, since high levels of PD-L1 are indicative of better clinical outcomes, it would be more meaningful to investigate cell lines with intermediate or low levels of PD-L1 (such as H1299 or A549) (Fig. 1a-b). Next, an in vivo sequential screening of cell lines was conducted to obtain a variant with higher metastatic capacity in Hu-PBMC mice, which can simulate recurrence and metastasis after surgery (Fig. 1c). However, subsequent in vivo experiments revealed that only subpopulation of H1299 (H1299-Luc) cell lines exhibited spontaneous metastasis approximately 1 week after subcutaneous excision, which are named as H1299M-Luc cells. In contrast, A549-Luc-derived xenografts failed to undergo lung metastasis (Fig. 1d-e). In addition to in vivo experiment, transwell assays were conducted to evaluate in vitro invasion ability of H1299M-Luc cells. The results indicated that H1299M-Luc cells displayed a greater propensity for metastasis compared to their parental cell line (Fig. 1f). In summary, we have generated a variant of human NSCLC cell line that can mimic postoperative recurrence and metastasis in humanized mice, thus providing an ideal platform for subsequent neoadjuvant immunotherapy experiments.
3.2 Neoadjuvant combination therapy can eradicate recurrence and metastasis
To assess the effectiveness of neoadjuvant immunotherapy alone or in combination with chemotherapy, we implanted H1299M-Luc cells into humanized mice and initiated neoadjuvant therapy once the tumor volume reached 150–200 mm3. Following six days of neoadjuvant therapy (either a combination of pembrolizumab and cisplatin or single-agent therapy), complete resection of subcutaneous tumors was performed in mice (Fig. 2a). The in vivo imaging system (IVIS) was utilized for postoperative monitoring of recurrence and metastasis in mice. We observed that neoadjuvant pembrolizumab monotherapy significantly suppressed tumor growth, and its antitumor efficacy was further potentiated by combination with cisplatin. Notably, neoadjuvant cisplatin monotherapy exhibited limited efficacy in inhibiting tumor growth (Fig. 2b). At the conclusion of the study, tumor tissues were collected, quantified, and visually documented. These findings further substantiate the superior anti-tumor efficacy of pembrolizumab in combination with cisplatin as a neoadjuvant therapy (Additional file 1: Fig. S2). Next, we assessed the impact of neoadjuvant therapy on recurrence and metastasis using IVIS at postoperative days 3, 6, 9, 12, and 15. The result showed that mice in the neoadjuvant combination group exhibited the lowest burden of recurrence and metastasis among four groups. Consistent with the trend in tumor growth, neoadjuvant pembrolizumab monotherapy could also improve the prognosis of mice when compared to the control group. Notably, the neoadjuvant chemotherapy exhibited limited benefit in the prognosis of mice, which is consistent with its limited efficacy in clinical practice. Further quantitative analysis of fluorescence confirmed the aforementioned trend (Fig. 2c-d)(Additional file 2: Table S1). Similarly, the IVIS results revealed a lower incidence of metastases in lung and liver of mice treated with neoadjuvant pembrolizumab or combination therapy (Fig. 2e). Further, H&E staining of lung and liver tissues was performed to determine metastasis in organs within each group more accurately. Similarly, the neoadjuvant pembrolizumab and combination groups demonstrated a significant reduction in lung and liver metastases compared to the other two groups (Fig. 2f-i). Together, these findings collectively suggest the promising efficacy of combined immunotherapy and chemotherapy in neoadjuvant settings.
3.3 Neoadjuvant therapy can reprogram T cell subsets both in tumor and peripheral blood
We investigated the underlying mechanism by which neoadjuvant chemoimmunotherapy improves prognosis. Given previous studies indicating the critical role of TILs in immunotherapy efficacy, we performed immunofluorescence analysis to examine CD45+ cell infiltration and distribution within primary tumors. Infiltration of immune cells in the neoadjuvant pembrolizumab and neoadjuvant combination group resulted in a shift towards an immune-inflammed phenotype (Fig. 3a). Quantitative analysis also revealed a significant increase in the proportion of total CD45+ cells in both treatment groups (Fig. 3b).
Considering the pivotal function of T cells in antitumor immunity, we evaluated the infiltration of primary T cell subsets (CD8+ T cells and CD4+ T cells) within primary tumor. Neoadjuvant pembrolizumab and combination therapies significantly increased CD8+ T cell infiltration within tumors compared to the other two groups (Fig. 3c). However, no significant difference was observed in terms of CD4+ T cells infiltration among treatment cohorts (Fig. 3d). Next, in order to identify the specific subtypes of CD8+ T cells that respond to neoadjuvant therapy, we conducted further analysis on the activation of CD8+ T cells. Representative images and relative quantification of CD8+PD-1+ T cells and CD8+granzymeB+ T cells were obtained through immunofluorescence (Fig. 3e-f, Additional file 1: Fig. S3A). An Increased proportion of CD8+PD-1+ T cells and CD8+granzymeB+ T cells were observed in both groups, indicating the activation of CD8+ T cells (Additional file 1: Fig. S3B). These findings suggest that neoadjuvant chemoimmunotherapy could effectively reprogrammed the TIME.
In addition to the primary tumor, investigating the effects of neoadjuvant therapy on peripheral immunity (such as peripheral blood and spleen) is warranted. We conducted an analysis of CD45+CD8+ T cell frequency and CD8+PD-1+ T cell frequency in both blood and spleen. Our findings indicate that neoadjuvant combination therapy led to increased activation and frequency of CD8+ T cells in both peripheral blood (Fig. 3g) and spleen (Fig. 3h). These findings suggest that neoadjuvant chemoimmunotherapy can improve the immune microenvironment both in the TME and peripheral immunity.
Finally, in order to ascertain whether immune cell reprogramming occurs during tumor metastasis, we conducted an analysis of the CD8+ T cell count in liver and lung metastases. However, no significant differences were observed among treatment groups with respect to the number of CD8 + T cells present within the metastatic lesions (Fig. 3i). Notably, although neoadjuvant chemoimmunotherapy did not alter the immune microenvironment of metastasis, it significantly suppressed tumor cell proliferation as evidenced by reduced ki-67 expression (Additional file 1: Fig. S4). Therefore, neoadjuvant chemoimmunotherapy has the potential to reprogram CD8+ T cells both in the tumor and periphery.
3.4 CD8+ T cells determine the efficacy of neoadjuvant chemoimmunotherapy
Given the significant alterations observed in CD8+ T cells within both the TME and peripheral immunity following neoadjuvant combination therapy, it is imperative to further investigate whether CD8+ T cells represent a pivotal immune subset for preventing recurrence and metastasis. To this end, human CD8+ T cells in Hu-PBMC mice were depleted by adminstered with in vivo CD8+ T cell antibodies to further test the efficacy of neoadjuvant combination therapy (Fig. 4a). The results of flow cytometry and immunohistochemistry demonstrated that in vivo administration of CD8+ T cell antibodies effectively depleted CD8+ T cells while preserving other immune cell subsets (Fig. 4b, Additional file 1: Fig. S5). Subsequent monitoring of tumor volume revealed that the neoadjuvant combination therapy lost its efficacy upon depletion of CD8+ T cells (Fig. 4c). Similarly, the IVIS results demonstrated that neoadjuvant combination therapy's effect was nullified by administering anti-CD8+ T cell antibodies, resulting in severe recurrence and metastasis similar to that of the control group (Fig. 4d-e). These findings suggest that CD8+ T cells are crucial for inhibiting local tumor growth and preventing recurrence and metastasis.
To further investigate the association between neoadjuvant immunotherapy efficacy and CD8+ T cells, we examined their correlation with liver and lung metastasis in mice. Our findings indicate that intratumoral infiltration of CD8+ T cells is inversely associated with the number of liver and lung metastases (Fig. 4f). Similarly, peripheral CD8+ T cells also exhibit a negative correlation with the incidence of liver and lung metastases. (Fig. 4g). The results of the correlation analyses indicated that both intratumoral and peripheral CD8+ T cells were significantly associated with mice prognosis, suggesting their potential as biomarkers for neoadjuvant therapy. Given the invasiveness of tumor biopsy and its inability to be repeated, we opted to employ more convenient peripheral blood CD8+ T cells as a viable alternative for subsequent verification.
3.5 Peripheral CD8+PD-1+ T cells are a biomarker of neoadjuvant therapy in preclinical model
An ideal biomarker for neoadjuvant therapy should reflect the dynamic changes in the host's anti-tumor immune response in real-time, thereby aiding in determining the ideal timing of surgery. Given that peripheral blood CD8+ T cells have been identified as a potential biomarker, it is imperative to validate whether their highest levels correspond to optimal local anti-tumor response and surgical timing. As shown in Fig. 5a, mice were euthanized at 0, 2, and 4 days post-therapy (designated as D0, D2, and D4 groups respectively) to investigate the kinetics of biomarker (peripheral CD8+ T cells) and document its peak level. The results of flow cytometry demonstrated that at the completion of therapy (D0 group), the neoadjuvant chemoimmunotherapy group exhibited a higher count of CD45+CD8+ T cells in peripheral blood compared to the control group. Furthermore, the levels of peripheral CD45+CD8+ T cells exhibited a curvilinear pattern and reached their peak on 2 days post-treatment (D2 group), indicating that the potential biomarker (peripheral CD8+ T cells) attained its maximum level two days after cessation of therapy. We also monitored the kinetics of PD-1-expressing CD8+ T cells in peripheral blood, which are generally recognized as tumor-specific T cells and crucial for the long-term survival of early-stage NSCLC patients. Similarly, the level of peripheral CD8+PD-1+ T cells exhibited a curvilinear change and reached its peak on the D2 group (Fig. 5b). In addition, bioinformatics analysis also indicates a significant positive correlation between the abundance of CD8 + PD-1 + T cells within the tumor immune microenvironment and survival in early-stage non-small cell lung cancer patients (Fig. 5c). Together, these dynamic changes in peripheral blood suggests that the D2 group may correspond to the optimal anti-tumor immune response.
To this end, we further confirmed the infiltration of CD8+ T cells or CD8+PD-1+ T cells within the TIME in the aforementioned groups, as well as assessed tumor necrosis and proliferation. The results indicated that mice in the D2 group exhibited significantly higher levels of both CD8+ T cells and CD8+PD-1+ T cells compared to those in the D0 and D4 groups, suggesting a close association between changes in peripheral biomarkers and reprogramming of the TIME (Fig. 5d). Additionally, given the pivotal role of tumor pathological response in clinical neoadjuvant immunotherapy and surgery, we have also investigated the necrosis and proliferation of primary tumors at various time points following treatment. The three neoadjuvant therapy groups demonstrated significant tumor necrosis and decreased Ki67 expression compared to the control group. Among them, the D2 group exhibited the largest necrotic area and lowest proliferation when compared to those of D0 and D4 (Fig. 5e-f). These findings suggest that the kinetics of peripheral CD8+PD-1+ T cells may serve as a predictive indicator for pathological responses and TIME reprogramming.
Finally, to definitively confirm the predictive ability of this biomarker for survival outcome of mice, we reconstructed a batch of mice and performed surgery at 0, 2, and 4 days post-treatment cessation. The IVIS imaging system was utilized to evaluate the impact of varying surgical intervals on the prognosis of neoadjuvant chemoimmunotherapy. Compared with the control group (treatment with saline), all neoadjuvant therapy (surgery at day 0, day 2, and day 4) were effective in eliminating tumor recurrence. Notably, mice that underwent surgery two days after therapy (D2 surgery) did not develop any metastases, indicating the most dramatic improvement in prognosis (Fig. 5g). Additionally, survival analysis was conducted on the mice in each group. All mice were euthanized 21 days after surgery, during which time there remained more than 25% hCD45+CD3+ T cells in peripheral (data not shown). Mice in the D2 surgery group (10/12) displayed longer survival times than those in the D0 (7/12) and D4 (5/12) groups (Fig. 5h). These finding strongly indicate that peripheral CD8+PD-1+ T cells serve as ideal biomarkers for neoadjuvant therapy. Their dynamic fluctuations can accurately predict the reprogramming of TIME and, more importantly, determine the optimal timing for surgery.
Since the kinetics of CD8+PD-1+ T cells in peripheral blood can serve as a predictor for survival outcome following neoadjuvant therapy, it is imperative to further investigate the potential interaction between its dynamic changes and surgical intervention. Therefore, we investigated the impact of different surgical timing on biomarkers by dynamically monitoring CD8+PD-1+ T cells in mice blood (Fig. 5i). We observed that premature surgical resection (D0 surgery) could impede the expansion of peripheral CD8+PD-1+ T cells following neoadjuvant therapy, whereas when surgical resection was too late (D4 surgery), the peripheral immune response had already declined before surgery. At the appropriate time of surgery (D2 surgery), the peripheral CD8+PD-1+ T cells of mice were maintained at the highest level before and after surgery. Notably, surgical resection at each time can cause a rapid decline in peripheral immunity; this immunosuppressive state may be attributed to the trauma and blood loss incurred during surgical resection.
Collectively, these results reveal a strong link between peripheral CD8+PD-1+ T cells, TIME, tumor cell proliferation, and prognosis in mice and identify peripheral CD8+PD-1+ T cells as a convenient biomarker for neoadjuvant immunotherapy.
3.6 Peripheral CD8+PD-1+ T cells in patients can predict both pathologic response and TIME
To further validate the predictive efficacy of peripheral CD8+PD-1+ T cells in clinical setting, blood and tumor samples were collected from 14 NSCLC patients (stage ⅠA to ⅢB) undergoing neoadjuvant chemoimmunotherapy (Additional file 3: Table S2). Specifically, blood samples were obtained from patients before and during neoadjuvant therapy, while tumor specimens were collected post-surgery. There were no unexpected adverse events observed during therapy. First, we assessed the pathologic response of tumor specimens from14 patients. The result of histologic assessment showed that 10 of 14 patients had a complete (no residual tumor identified; n = 8) or major (10% or less viable tumor cells; n = 2) pathologic response (Fig. 6a). In addition, the dynamic monitoring results of peripheral blood demonstrated a significant increase in CD8+PD-1+ T cells among most patients following the neoadjuvant chemoimmunotherapy, which is consistent with the findings in preclinical models (Fig. 6b). Given that pathological responses are now widely utilized as predictors of long-term survival in neoadjuvant clinical trials, it is imperative to examine the correlation between our proposed novel biomarkers and these responses. As depicted in Fig. 6c, the frequency of peripheral CD8+PD-1+ T cells was significantly higher in patients with complete or major pathologic response compared to those without any apparent pathological response. This indicates that the peripheral CD8+PD-1+ T cells can predict pathological responses in patients receiving neoadjuvant therapy.
Next, to further validate the correlation between pathological responses and TILs, we employed immunofluorescence to examine the infiltration and activation of CD8+ T cells in tumor exhibiting diverse pathological responses (Fig. 6d). The results revealed that tumors without apparent pathological response displayed minimal infiltration of CD8+ T cells and CD8+PD-1+ T cells, which was comparable to the level observed in pre-treatment biopsies. Notably, tumors with MPR and CPR exhibited significantly increased infiltration of both CD8+ T cells and CD8+PD-1+ T cells, with the highest levels observed in CPR tumors (Fig. 6e). This finding suggests that tumor with optimal pathological response is typically accompanied by an immuno-inflamed phenotype. Since previous studies have reported that immuno-inflamed tumor could predict the long-term survival in patients with advanced NSCLC, we conducted an analysis to investigate the correlation between peripheral CD8+PD-1+ T cells and immune cell infiltration within tumor. Correlation analysis revealed a positive association between peripheral CD8+PD-1+ T cells and immune cell infiltration in the tumor microenvironment (Fig. 6f). Overall, the kinetics of peripheral CD8+PD-1+ T cells in patients can serve as a reliable predictor for both pathological response and reprogramming of TIME.
3.7 Reprogramming of TIME in patients receiving neoadjuvant chemoimmunotherapy
To investigate the impact of neoadjuvant therapy on other immune cell subsets within TIME, we examined a subset of 2 patients who had early imaging and paired pre- and post-treatment tumor samples. A 57-year-old man was diagnosed with left lower lobe squamous cell carcinoma (cT4N1M0-stage IIIA) (Fig. 7a). The patient was administered nab-paclitaxel at a dose of 200 mg on day 1 and day 8, cisplatin at a dose of 500 mg on day 1, and pembrolizumab at a dose of 200 mg on day 2. After two cycles of therapy, we conducted a CT reassessment and observed a reduction in the patient's lesion to stage IIB. Concurrently, there was significant relief of lesions near the fissure, accompanied by remission at the basal trunk of the left lower lung and left lower pulmonary vein. Notably, the resection of the tumor resulted in an MPR (residual active tumor cells < 10%), which is a significant biomarker for long-term survival in patients undergoing neoadjuvant immunotherapy (Fig. 7b). Furthermore, we performed multiple fluorescence analyses on tumor samples pre- and post-treatment (Fig. 7c). Similar to the aforementioned preclinical findings, a significant augmentation in CD8+ T cell count was observed following neoadjuvant therapy. Additionally, an increased proportion of CD8+PD-1+ T cells and CD8+GrB+ T cells were noted. Most notably, neoadjuvant therapy resulted in a substantial reduction in the number of Treg cells (Foxp3+ T cells and Foxp3+PD-1+ T cells) (Fig. 7d).
In another case, a 59-year-old male patient was diagnosed with left upper lobe squamous cell carcinoma (cT1N3M0-stage IIIB). Following two cycles of neoadjuvant combination therapy (pembrolizumab 200 mg day 1 + docetaxel 120 mg day 1 + cisplatin 120 mg day 1), the metabolic profile of the lesion was reassessed using PET-CT. Imaging results indicated that the tumor had been downstaged to ycT1bN0M0 (stage Ia2) (Additional file 1: Fig. S6A). After 38 days following the completion of neoadjuvant therapy, surgery was performed and the specimens exhibited a pCR (Additional file 1: Fig. S6B). In contrast to the aforementioned patient, this individual underwent two additional cycles of adjuvant immunotherapy post-surgery. At the 17-month follow-up, no instances of tumor metastasis or recurrence were reported. Similarly, immunofluorescence analysis of specimens revealed a significant infiltration of CD45+ immune cells and a substantial presence of CD8+PD-1+ T cells and CD8+GrB+ T cells (Additional file 1: Fig. S7). Taken together, these findings suggest that neoadjuvant chemoimmunotherapy can improve the TIME of resectable NSCLC patients.