HOXC9 characterizes a suppressive tumor immune microenvironment and integration with multiple immune biomarkers predicts response to PD-1 blockade plus chemotherapy in lung adenocarcinoma

Gene expression analysis

We employed the online tools in the ‘Exploration’ mode of Tumor Immune Estimation Resource 2.0 (TIMER2) (http://timer.cistrome.org/) [20] to acquire diverse expression patterns of HOXC9 across various cancers and neighboring healthy tissues. The raw data underwent normalization through log2(TPM) (Transcripts Per Kilobase of exon model per Million mapped reads) conversion.

Genetic alteration and promoter

The estimation of HOXC9 genetic changes was conducted using the web-based cBioPortal repository (https://www.cbioportal.org/) [21, 22]. This analysis relied on data from the TCGA pan-cancer atlas research database [23]. The mutation distribution of HOXC9 was graphed using the Catalogue of Somatic Mutations (COSMIC) (http://www.sanger.ac.uk/cosmic/) [24], a publicly available resource that provides details about somatic mutations found in human cancers.

Survival prognosis analysis

Visualized through R’s survival and survminer packages, the Kaplan-Meier assessment of LUAD’s HOXC9, derived from the TCGA database alongside clinical data from our hospital, was conducted. The computed values encompassed the log-rank P-value, hazard ratio (HR), and 95% confidence intervals (CI).

Cells and reagents

The CMT167 and Lewis lung cancer (LLC) cell lines derived from mice were procured from European Collection of Authenticated Cell Cultures (ECACC), and the American Type Culture Collection (ATCC). They were nurtured in DMEM medium (Gibco, Thermo Fisher Scientific, Inc., USA), supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) and 1% Penicillin-Streptomycin (Solarbio, Beijing, China). Incubation was carried out in a 37° C environment with 5% CO₂ and 95% humidity. Following procurement, the cell lines underwent authentication through Short Tandem Repeat (STR) analysis, and routine screenings verified their negative status for mycoplasma contamination.

Hoxc9 overexpression and knockdown construction

In order to create knockdown of Hoxc9 cells, two small interfering RNA (siRNA) molecules targeting Hoxc9 (siHoxc9-1 and siHoxc9-2), along with a control siRNA (siCtrl), were generated and synthesized by Shanghai GenePharma Co. The initial step involved seeding lung cancer cells into six-well plates, proceeding with transfection when cell density reached 80–90% confluence. Lipofectamine3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was employed to transfect CMT167 and LLC cells with siRNAs or siCtrl, following the manufacturer’s provided protocols. The antisense sequences corresponding to the three siRNAs were displayed as follows:

siHoxc9-1: 5′- CAGTCGTCTGTGGTCTATCACCCTT-3′; siHoxc9-2: 5′- AGTCGTCTGTGGTCTATCACCCTTA-3′; siCtrl: 5′-CAGCTGTGTTGGATCACTCCTCCTT-3′.

Immunoblotting and qPCR techniques were utilized to confirm gene expression levels. The sequence of siHoxc9-1 informed the creation of shRNA constructs. These constructs, along with lentivirus packaging vectors, were introduced into HEK-293T cells, facilitating the generation of lentiviral particles for either shRNA-mediated gene silencing or Hoxc9 overexpression. The produced viral particles, with a multiplicity of infection (MOI) set at 20, were purified and then applied to cancer cells. Subsequent to viral transduction, puromycin was employed as a selective agent to establish cell lines stably expressing either silenced or overexpressed Hoxc9.

Real time quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated from cells 48 hours post transfection utilizing TRIzol reagent (Invitrogen, USA). The sample loading scheme was determined based on the count of gene samples and cells examined. To conform this scheme, a mixture was prepared by combining iTaqUniversal SYBR RT-qPCR mix reagent (Bio-Rad, USA) and primers. All steps in the experimentation adhered to the manufacturers’ guidelines. RT-qPCR was employed to identify the relative expression of Hoxc9 within CMT167 and LLC cells. Hoxc9-specific primers were used for PCR amplification, designed as per the subsequent sequences:

Forward: 5’-CCGACCTGGACCCTAGCAAC-3’.

Reverse: 5’-CCGACGGTCCCTGGTTAAAT-3’.

Actb-specific primers were used for PCR amplification, designed as per the subsequent sequences:

Forward: 5’-GTGACGTTGACATCCGTAAAGA-3’

Reverse: 5’-GCCGGACTCATCGTACTCC-3’

An RT-qPCR apparatus (Roche, USA) was employed to initiate the reaction with a programmed setup. To each well, 1μL of cDNA template was subsequently introduced. Following this, the experiments were conducted in triplicate. The qPCR data was obtained by exporting the results, and the data’s reliability was assessed and examined based on the melting curve analysis.

Western-blot

To determine Hoxc9 protein expression, Western blotting technique was employed. Initially, RIPA lysis buffer was used to extract total proteins from CMT167 and LLC cells. Protein quantification was carried out using the BCA Protein Assay Kit (Biovision, USA). For protein electrophoresis, a Western-Blotting electrophoresis system (Bio-Rad, USA) was utilized, with 20 micrograms of protein loaded per well. Subsequently, transfer onto a PVDF membrane (Bio-Rad) was executed. Following a blocking phase with 10% skim milk, the membranes underwent antigen-antibody interactions, employing specific antibodies including anti-DDK-tag monoclonal antibody (1:1000, sourced from Abcam), anti-Hoxc9 antibody (1:1000, obtained from Absin), and β-actin (diluted 1:2000; provided by CST). This binding sequence was conducted under persistent shaking, maintained throughout the night at 4° C. Subsequently, on the following day, the membranes were treated with appropriate horseradish peroxidase-conjugated secondary antibodies, specific to each species (diluted 1:5000), and incubated for two hours at ambient temperature. The final visualization of the bands was accomplished using a chemiluminescence detection system.

Cell viability assay

Cell viability assessment was conducted using the Cell Counting Kit-8 (Takara, China). A cell suspension containing CMT167 and LLC cells in robust growth conditions was prepared, achieving a concentration of 2×10³ cells per 100 μl. Subsequently, 100 μL of the cell suspension was dispensed into individual wells of a 96-well plate and positioned within an incubator set at 37° C for initial incubation.

Following this, the subsequent day involved the introduction of 10 μL of CCK-8 solution into each well of a 96-well plate, which was followed by the removal of the preceding culture medium. After undergoing incubation for a duration of 2 hours, the absorbance (OD) was gauged at a wavelength of 450nm using the Multiscan FC microplate reader. The entire procedure was replicated thrice to ensure reliability and consistency.

Colony formation assay

The evaluation of lung cancer cell proliferation resulting from Hoxc9 knockdown or overexpression was performed using the colony formation assay. Post transfection, CMT167 and LLC cells were digested and then planted into 6-well plates with a cell density of 500 cells per well, in accordance with the characteristics of lung cancer cells. Subsequently, they were placed in a 37° C incubator with 5% CO₂ and subjected to culturing. When visible clones emerged in most cells within the six-well plate after 10 days of culturing, the culturing process was halted. Following this, formaldehyde was employed for fixation lasting 15 minutes, succeeded by staining with crystal violet for a duration of 10 minutes. Utilizing camera software, images of the clone wells were captured, and clone counting was executed through ImageJ software. Each group underwent a triplicate evaluation of the wells.

Wound healing assay

Seeded into a 6-well plate were CMT167 and LLC cells. Upon reaching 80% confluence of the cells, the monolayer cells were gently scratched using a sterile micropipette tip. The floating cells were subsequently cleansed with PBS. At 0 hours and 24 hours, the process of wound healing within the scratch was observed. Each condition was evaluated in sets of three wells.

Cell invasion assay

To conduct the cell invasion assay, 24-well plates with chambers featuring an 8-μm pore size (Corning, Inc.) were utilized. Matrigel was diluted using serum-free medium at a ratio of 1:8. The resultant matrix gel was uniformly applied onto the membrane situated at the base of the Transwell chamber. This assembly was then placed within a 37° C incubator for an incubation period of 2 hours. Following a 48-hour transfection, the lung cancer cells suspension was formulated using serum-free culture medium, with a subsequent adjustment of cell density to 5×10⁵/ml. Simultaneously, 5×10⁴ cells were seeded into each well of the Transwell chamber. In the lower compartment of the 24-well plate, 500-650μL of medium containing 10% FBS was typically introduced. Ultimately, the Transwell chamber was removed, fixed with 4% paraformaldehyde, and subsequently stained using 0.5% crystal violet. Cells present on the upper surface of the filter membrane were gently eliminated using a cotton swab. Employing an optical microscopy, images of cells traversing the invasion chamber were captured.

Tumor-bearing murine models and treatments

To establish tumor-bearing murine models, 1.0 × 10⁶ LLC cells (infected with lentiviral particle) in 200uL PBS were implanted subcutaneously in the right hind flank of syngeneic BALB/c mice at day 0. Tumor growth was examined at 2, 5, 8, 11, 14, 17 and 20 days. On day 6, 36 mice were randomly assigned to each group, control with IgG isotype (N=6), control with anti-PD-1 (N=6), over expression of Hoxc9 with IgG (N=6), over expression of Hoxc9 with anti-PD-1 (N=6), knockdown of Hoxc9 with IgG (N=6) and knockdown of Hoxc9 with anti-PD-1(N=6). For the groups with anti-PD-1 therapy, an anti-PD-1 mAb (#BE0146, clone: RMP1-14) was administered i.p. every three days, starting at day 5. The control group was injected with IgG control (#BE0089, clone: 2A3) on each injection day. Tumor size was measured by digital calipers every three days and tumor volume was calculated as length×width²/2 (mm³). Mice were sacrificed and tissues were analyzed at or before the ethical tumor volume limit of 1000 mm³.

Flow cytometry for cell cycle, apoptosis and infiltrated immune cells analyses

To assess the impact of Hoxc9 knockdown on the cell cycle of CMT167 and LLC, flow cytometry analysis was conducted. CMT167 and LLC cells were trypsinized and gathered through centrifugation at 1000rpm for 5 minutes following 48 hours of siHoxc9 or siCtrl transfection. Subsequently, the fixed cells were suspended in pre-chilled 75% ethanol and placed in a refrigerator at 4° C overnight. Adhering to the guidelines from the flow cell cycle kit (Biyuntian, Shanghai), the cells were thoroughly mixed, then filtered through the membrane and transferred to a 5ml flow tube.

1X Binding Buffer was prepared according to the instructions for flow cytometry. Collect transfected lung cancer cells and resuspend the cells with 1X staining buffer to a cell concentration of 5 x 10⁶ cells/mL. Add 5μL fluorescent dye-conjugated Annexin V and incubate for 10-15 min at room temperature. The cells were then resuspended in staining buffer. 5μL propidium iodide (PI) dye was added and incubated on ice for 15 minutes. Flow cytometry was used to read the data.

Mice were sacrificed by cervical dislocation, and tumors were harvested to prepare single cell suspensions. The single-cell suspensions of tumor tissue were generated with tumor dissociation kit, mouse (Miltenyi Biotec, Germany) according to manufacturer’s instructions. Single cell suspensions were stained with fixable viability dye (Fixable Viability Stain 780, BD Pharmingen, USA) at room temperature for 10 minutes to rule out dead cells. Cell surface markers were assessed using the following antibodies incubated with cell suspensions in the dark at 4° C for 30 minutes: CD45 (BD Pharmingen), CD3 (1:20; #100205, #100218, Biolegend), CD4 BV605 (BD Pharmingen), IFN-γ (1:20; #505808, Biolegend). The flow cytometry data were acquired on Beckman Cytoflex (Beckman Coulter, USA) and the results were analyzed with FlowJo V.10.6.1 software (TreeStar).

Study population, treatment and assessments

Prospective enrollment took place at Shanghai Pulmonary Hospital, involving patients with advanced LUAD. Inclusion criteria encompassed age between 18 and 70, confirmed histologically or cytologically as stage IV LUAD (as per the 8th edition of the International Association for the Study of Lung Cancer Staging Handbook in Thoracic Oncology), lack of EGFR and ALK alterations, ECOG PS score of 0 or 1, no prior systemic therapy, presence of at least one measurable lesion following RECIST v1.1, and a projected life expectancy of over 3 months. Complete baseline characteristics or radiologic images, including chest CT, brain MRI, and PET-CT scan, were evaluated by two specialized radiologists. Before commencing the first-line combination of PD-1 blockade and chemotherapy, tumor samples were acquired through image-guided percutaneous lung biopsy. PD-L1 expression was evaluated as part of the initial screening procedure at the central pathology laboratory within our institution. This evaluation hinged on the application of the Tumor Proportion Score (TPS), a metric that gauges the proportion of viable tumor cells displaying partial or complete membrane staining, regardless of the intensity of staining. The resulting values from this assessment were routinely classified into three distinct groups: TPS <1%, TPS 1-49%, and TPS ≥50%. The study was approved by the ethics committee of Shanghai Pulmonary Hospital and was conducted in accordance with the Declaration of Helsinki (as revised in 2013). All participants signed informed consent forms before the initiation of this research undertaking.

Treatment was administered until radiographic progression, the occurrence of intolerable toxic effects, a determination made by the investigator, or the voluntary withdrawal of consent by the patient. If toxicity could be definitively attributed to a single agent, that specific drug could be discontinued. Tumor imaging sessions were planned at weeks 6 and 12, followed by intervals of 9 weeks until week 48, and subsequently, at 12-week intervals. The assessment of response was conducted in accordance with RECIST, version 1.1 [25]. Additionally, there was a regular patient contact every 12 weeks to evaluate survival outcomes during the follow-up period. The primary endpoints encompassed OS and PFS. OS indicated the time from randomization to death resulting from any cause. PFS denoted the time from randomization to either disease progression or death, whichever transpired first, as evaluated by a blinded, independent central radiologic review. Secondary endpoints included the response, the duration of response, and safety.

Immunohistochemical staining

Paraffin-embedded lung cancer tissue samples were sliced to 4um thickness and affixed onto slides for subsequent deparaffinized hydration. The tissue was then restored to its postfixed state, exposing the antigen for precise antibody binding. Following antigen restoration under high temperature and pressure, endogenous peroxidase was neutralized through a 10-minute treatment with hydrogen peroxide. Blocking utilized goat-derived animal serum. Antibodies were incubated with sections before immunohistochemical (IHC) staining. The slices were covered with diluted HOXC9, CD4, CD8, CD68 and FOXP3 (Abcam, Cambridge, MA, USA) mouse monoclonal antibodies, and this covering occurred in a humidified chamber for 2 hours at 37° C. After an overnight 4° C block, sections were fully covered with a secondary antibody and incubated at 37° C for 30 minutes. Subsequent DAB (Abcam, Cambridge, MA, USA) coloration was meticulously controlled. Slices were sealed with neutral gum, air-dried in a fume hood, and then microscopically analyzed for images. Staining assessments were performed simultaneously by two independent observers, scoring each stained section and reaching a consensus. According to the median percentage of positive cell proportion in each marker, the cut-off values of positive status were calculated.

Statistical analysis

Statistical analyses were performed using the aforementioned online databases and R software (version 3.6.3). Data are presented as mean ± SD. For the statistical analysis of experimental data, GraphPad Prism 9.5.1 (GraphPad Software; Dotmatics) was employed. Unpaired Student’s t test and Wilcoxon rank-sum test and one-way ANOVA tests (with LSD as post hoc test) were used to calculate the significance of differences in data between and among groups. P<0.05 was considered to indicate a statistically significant result.

Availability of data and materials

Bioinformatics datasets presented in this study can be found in online repositories, and the datasets used and/or analyzed during experiments are available from the corresponding author on reasonable request.

Consent for publication

All authors have approved the manuscript for submission.

HOXC9 is overexpressed and correlated with prognosis in LUAD

Firstly, we assessed HOXC9 expression in pan-cancer data from TCGA and GTEx. The results showed that HOXC9 was significantly higher in 14 types of tumors than corresponding normal tissues including BLCA, BRCA, CHOL, ESCA, GBM, HNSC, LUAD, LUSC, PCPG, PRAD, SKCM, STAD, THCA, and UCEC, with TIMER2 pan-cancer analysis (all P<0.05) (Figure 1A). Then, the correlations between HOXC9 expression level and different clinical variables in LUAD were analyzed via UALCAN tools, which indicated higher HOXC9 expression was significantly associated with higher individual stages and tumor grade (Figure 1B–1E). To confirm this finding, we compared HOXC9 protein level between adjacent normal and cancerous tissues from clinical LUAD samples, and also found it was upregulated in tumor sample (Figure 1F, 1G). Moreover, the receiver operating curve, with the aera under the curve of 0.741 (95%CI 0.691-0.791), indicated a high diagnostic value of HOXC9 in LUAD, as is shown in Figure 1H. A survival analysis was performed, showing high expression of HOXC9 was associated with shorter OS (HR=1.52, 95%CI 1.140-2.030, P=0.001) in LUAD (Figure 1I).

Figure 1. Analyses of HOXC9 expression and the diagnostic and prognostic value in LUAD. (A) The expression distribution of HOXC9 in tumor tissues and normal tissues with TIMER2, respectively; (B–E) The correlation between HOXC9 expression and clinical variables of LUAD; (F) The HOXC9 protein level in adjacent healthy tissue; (G) and in the tumor tissue; (H) The diagnostic value of HOXC9 in LUAD; (I) The OS analysis of HOXC9 in LUAD patients. (*, P<0.05; **, P<0.01; ***, P<0.001).

At the facet of genomic exploration, through the online database cBioPortal, HOXC9 genetic mutations and alterations landscape were investigated in various tumor samples from TCGA datasets (Supplementary Figure 1A). The main type of genetic alterations in HOXC9 was “mutation”, which were observed in bulk of TCGA cancers, and the “amplification” was the second most common. The frequency of HOXC9 alteration in LUAD patients was mRNA high. With COSMIC online tool, the overview of the types of mutation was observed. The primary mutation type was missense substitution (52.43%), and the primary substitution mutation type was C>T (33.22%) (Supplementary Figure 1B).

HOXC9 expression is related to repressive immune cells and play a hazard role in responses to immunotherapy

The relationship between HOXC9 expression and immune repressive cells’ infiltration (myeloid-derived suppressor cells and Tregs) was explored in Figure 2A, 2B. To further explore the role of HOXC9 in TME, we conducted a correlation analysis of HOXC9 expression with other immune infiltration cells in LUAD, which demonstrated that patients with relatively high HOXC9 possessed less dendritic cells and T central memory cells (Figure 2C). To further verify the negative role of HOXC9 in responses to immunotherapy, the data from TIDE showed relatively higher HOXC9 was related to worse OS in bladder cancer (P=0.043, Figure 2D), kidney cancer (P=0.027, Figure 2E), and melanoma (P=0.016, Figure 2F) treated with ICIs.

Figure 2. The correlation analysis between HOXC9 level and suppressive immune cells among cancers. (A) HOXC9 was significantly related to MDSC in LUAD. (B) HOXC9 was significantly related to Tregs in LUAD. (C) patients with high HOXC9 possessing lower DC and Tcm infiltrations. (D) HOXC9 expression was related to shorter OS of bladder cancer. (E) kidney cancer. and (F) melanoma treated with ICIs. (*P<0.05, **P<0.01, ***P<0.001).

Knockdown of Hoxc9 reduces the proliferative capacity of in murine NSCLC cells

Taken together, HOXC9 is an oncogene that drives tumorigenesis and progression in lung cancer and can lead to poor prognosis by activating cell proliferation and causing immune dysfunction. We then performed a series of in vitro experiments to verify that knockdown of Hoxc9 at the cellular level could inhibit its malignant biological behavior. Subsequently, CMT167 and LLC cell lines were transfected with siRNA against Hoxc9. The knockdown efficiency of siHoxc9-1 and siHoxc9-2 was assessed by RT-qPCR, and the results showed that siRNA effectively reduced the mRNA level of Hoxc9 in CMT167 and LLC cells (Figure 3A, 3B). Subsequent western blot experiments also verified that the protein expression level of Hoxc9 was significantly reduced after transfection of siRNA (Figure 3C, 3D). CCK8 cell proliferation assay was used to detect the effect of knockdown of Hoxc9 on cell proliferation. Compared with siNC (si negative control) group, the proliferation rate of CMT167 and LLC cells in Hoxc9 knockdown group was significantly reduced (Figure 3E, 3F, P<0.001). Colony formation assay was used to detect the effect of Hoxc9 knockdown on cell colony formation. Compared with vector group, the number of CMT167 and LLC colony groups in Hoxc9 knockdown group was significantly reduced (Figure 3G, 3I, P<0.001). The results showed that knockdown of Hoxc9 expression reduced cell proliferation and significantly inhibited the colony formation ability of CMT167 and LLC cells.

Figure 3. Confirmation of Hoxc9 knockdown and its impacts on viability and proliferation of CMT167 and LCC cells. (A, B) The knockdown level of Hoxc9 was detected by RT-qPCR. (C, D) The knockdown level of Hoxc9 was detected by western blot. (E, F) The cell viability was significantly impaired under Hoxc9 knockdown. (G–I) The influenced proliferation competence was detected with colony formation after 10 days. (*P<0.05, **P<0.01, ***P<0.001).

Knockdown of Hoxc9 promoted apoptosis and cell cycle arrest

Flow cytometry was used to analyze the effect of Hoxc9 knockdown on cell cycle and apoptosis. The results of cell cycle experiments suggested that the number of cells in S phase were significantly increased and the number of cells in G1 phase was decreased in the two Hoxc9 knockdown groups compared with the siNC group (Figure 4A–4D, P<0.001), indicating that the proliferation ability was significantly impaired after blocking Hoxc9 in CMT167 and LLC cells. Subsequently, flow cytometry PI/Annexin V was used to analyze the effect of Hoxc9 knockdown on the apoptosis of CMT167 and LLC lung cancer cells. The results suggested that the number of early apoptotic cells was significantly increased in the two Hoxc9 knockdown groups compared with the siNC group (Figure 4E–4H, P<0.001). The results showed that knockdown of Hoxc9 expression caused lung cancer cell apoptosis, and this biological effect may be related to cell cycle S phase arrest.

Figure 4. Hoxc9 knockdown arrested cell cycle and promoted cell apoptosis. (A–D) Hoxc9 knockdown arrested cell cycle of CMT167 cells and LCC cells. (E–H) Hoxc9 knockdown promoted cell apoptosis of CMT167 cells and LCC cells. (*P<0.05, **P<0.01, ***P<0.001).

Knockdown of Hoxc9 reduces the migrative and invasive capacity of in murine NSCLC cells

As for the impacts of Hoxc9 knockdown on migration and invasion of CMT167 and LLC, we conducted the wound healing and Transwell assays to investigate. After 24h, cells transfected with siHoxc9 showed significantly wider remaining wound than siNC groups (Figure 5A–5D), and showed the evidently less penetrated cells than siNC groups (Figure 5E–5G).

Figure 5. Hoxc9 knockdown inhibited the migration and invasion of tumor cells. (A) Remaining length of wound of CMT167 under knockdown of Hoxc9 was significantly longer compared to control (200 μm). (B) Remaining length of wound of LLC under knockdown of Hoxc9 was significantly longer compared to control (200 μm). (C, D) The statistical analysis of above assays. (E–G) Count of migrated CMT167 and LCC cells was significantly decreased after knockdown of Hoxc9. (*P<0.05, **P<0.01, ***P<0.001) (100 μm).

Overexpression of Hoxc9 promoted cell proliferation and migration in lung cancer cell lines

The functional of Hoxc9 overexpression in lung cancer was determined by in vitro cell experiments. Ddk-tagged Hoxc9 overexpression vector was constructed and transfected into CMT167 and LLC cells. Western blot analysis confirmed that Hoxc9 was overexpressed in CMT167 and LLC cells after plasmid transfection (Figure 6A). Overexpression of Hoxc9 promoted lung cancer cell proliferation (Figure 6B, 6C) and colony formation (Figure 6D). In CMT167 and LLC cells, Hoxc9 overexpression promoted increased migration of lung cancer cells (Figure 6E, 6F) and invasion of lung cancer cells (Figure 6G). Overall, overexpression of Hoxc9 promotes lung cancer proliferation, colony formation, and tumor cell invasion and progression.

Figure 6. Overexpression of Hoxc9 elicited a promotive role in tumor cells progression. (A) Confirmation of Hoxc9 overexpression in two cell lines with WB. (B, C) Significantly higher cell viabilities were observed in the DDK-Hoxc9 groups of CMT and LCC. (D–F) Overexpression of Hoxc9 resulted in a more counts of colony and wider remaining wound (200 μm). (G) Overexpression of Hoxc9 resulted in more invasive counts of cells (100 μm). (*P<0.05, **P<0.01, ***P<0.001).

Differential Hoxc9 expression levels mattered tumor growth and CD8+ T cells infiltration with or without PD-1 blockade

With the establishment of tumor-bearing murine models, we verified the hypothesis that Hoxc9 expression level impacted tumor growth and sensitivity to PD-1 blockade, and the diagram was shown at Figure 7A. As shown in Figure 7B, a notable reduction in tumor growth rate was evident within the Hoxc9 knockdown group compared to the control group, both under IgG isotype conditions (P<0.05) and following PD-1 blockade intervention (P<0.01) (the tumor models photograph was in Supplementary Figure 2). Overall, while the efficacy of PD-1 blockade exhibited a positive trend over Hoxc9 knockdown, statistical significance was not achieved. Intriguingly, the overexpression of Hoxc9 nearly counteracted the effects of PD-1 blockade, as observed in the control group with IgG isotype. As anticipated, the most substantial disparity in tumor sizes was observed between the control group treated with IgG and the Hoxc9 knockdown group subjected to PD-1 blockade (P<0.001).

Figure 7. Combination of Hoxc9 inhibition and PD-1 blockade enhanced the tumor growth and improved CD8+ T cell infiltration. (A) Diagram of in vivo experiment design. (B) Tumor growth curves of different groups. (C, D) More effective CD8+ T cells were observed in Hoxc9 knockdown groups compared to control groups under IgG/PD-1 blockade. (*P<0.05, **P<0.01, ***P<0.001).

The present study involved the disaggregation of mouse tumor tissues into individual cells for subsequent flow cytometry analysis. The outcomes of the sorting process are illustrated in Figure 7C. In the isotype control group, there was no statistically significant augmentation observed in tumor-infiltrating CD3+ T cells. However, upon the attenuation of Hoxc9 expression, a noteworthy increase was noted in activated CD8+ T cells (P<0.05) and IFN-γ production (P<0.01) (Figure 7D). Conversely, the overexpression of Hoxc9 elicited an opposing effect. Additionally, with the treatment of PD-1 blockade, concurrent with tumor growth dynamics, the downregulation of Hoxc9 exhibited a pronounced augmentation in CD3+ T cell infiltration, activation of CD8+ T cells, and an upsurge in IFN-γ production (P<0.001). These findings collectively suggest a potential synergistic antitumor immune response through the concomitant modulation of immune checkpoints and Hoxc9.

HOXC9 expression improves the predictive power for response to PD-1 blockade plus chemotherapy and survival in LUAD

Three officially approved predictive biomarkers, including PD-L1 expression, TMB, and MSI, guide the clinical application of anti-PD-1/PD-L1 monotherapy with moderate predictive performance [3, 8]. However, effective predictive markers for ICIs combined with chemotherapy remain underreported [8, 9]. Within the TME, characterized by distinct subpopulations of immune cells such as CD3+, CD8+, CD68+, and FoxP3+ cells, as well as other known or potential molecules, exists a diverse array of predictors that warrants continuous exploration [3, 12]. To investigate the predictive value of HOXC9 expression in the context of PD-1 blockade plus chemotherapy, we prospectively enrolled advanced LUAD patients with no prior systemic treatment for first-line immunochemotherapy, and baseline biopsy samples were obtained for IHC analysis. Finally, from January 2021 to March 2021, 31 patients treated with anti-PD-1 plus chemotherapy from our center were included in the final analysis (Figure 8A). The baseline characteristics of the participants were presented in Supplementary Table 1. The positive and negative expression of HOXC9, CD4, CD8, CD68, and FOXP3 divided by the median were presented in Figure 8B. Our initial assessment involved examining correlations between individual TME marker expressions and objective responses. Notably, responders exhibited significantly higher CD8 positive expression, as indicated in Figure 8C. PD-L1 expression demonstrated an increasing trend among responders, the expressions of HOXC9 and FOXP3 displayed a decreasing trend, but statistically insignificant. Worth mentioning is the substantially low overall expression of FOXP3. The clinical practice widely employs a trichotomy of PD-L1 TPS using cutoff values of 1% and 50%. Unfortunately, this trichotomy’s predictive capacity for treatment response and survival proved to be even inferior to the dichotomy of PD-L1 TPS based on median values.

Figure 8. The predictive value of HOXC9 and TME markers for LUAD patients treated with PD-1 blockade plus chemotherapy. (A) Diagram of clinical samples selection and analysis. (B) IHC analysis of 5 proteins based on samples from 4 patients with different level of HOXC9. (C) The different distributions of responder population under 5 types of TME marker. (D) Analysis of predictive value of combination of HOXC9- and other markers. (E) PFS and OS analysis based on different status of markers. (F) PFS and OS analysis based on different combinations. (*P<0.05, **P<0.01, ***P<0.001).

We then conducted a focused investigation to examine the correlations between various combinations of HOXC9 and other markers and their impact on objective response. The results revealed a distinct differentiation between responders and non-responders, as illustrated in Figure 8D. Subsequently, we carried out a more detailed exploration into the prognostic value of individual markers and combinations of HOXC9 with other markers. Notably, none of the individual markers exhibited predictive ability for survival, as depicted in Figure 8E. However, patients characterized as PD-L1+/HOXC9- and CD8+/HOXC9- both showed a notably extended PFS (P=0.027 and P=0.030), as indicated in Figure 8F. In general, the inclusion of HOXC9 tended to enhance the differentiation of patients with differential prognosis. Consequently, the expression of HOXC9 generally enhances the predictive accuracy for both treatment response and survival outcomes when PD-1 blockade is combined with chemotherapy in cases of LUAD.