The tumor-associated microbiome is increasingly recognized as a critical modulator of cancer progression and therapy response, yet its composition and functional relevance in human lung tumors remain poorly defined. In this study, we profiled the bacterial and fungal communities of lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) and investigated how microbial patterns relate to tumor differentiation, immune contexture, and transcriptomic programs. LUAD and LUSC exhibited distinct microbial signatures, with LUAD showing lower microbial diversity and enrichment of genera associated with mucosal health. Within LUAD, differentiation status emerged as a key ecological and clinical variable: differentiated tumors were enriched in beneficial genera such as Akkermansia and Muribaculum, which correlated with CD8⁺ T cell infiltration and immune activation gene expression. In contrast, undifferentiated LUAD displayed increased abundance of pro-inflammatory or pathogenic taxa, including Lactococcus and Thermus, alongside reduced cytotoxic immune infiltration and elevated PD-L1 expression. Transcriptomic analysis revealed immune-related pathways in differentiated tumors and proliferative, MYC-driven programs in undifferentiated tumors. Integrated microbiota-transcriptome correlation and co-occurrence network analyses further delineated two distinct ecosystems: an immune-permissive, metabolically stable microbiota in differentiated LUAD versus a dysbiotic, immune-suppressive community in undifferentiated tumors. Functional predictions reinforced these trends, linking beneficial taxa to SCFA production and epithelial homeostasis, and harmful taxa to lipopolysaccharide biosynthesis and immune evasion. Collectively, these findings reveal that LUAD differentiation shapes the composition, structure, and function of the tumor microbiome, with implications for prognosis, immune responsiveness, and microbiota-informed therapeutic strategies.

Gallbladder carcinoma is a deadly disease with a poor prognosis, and recent clinical data suggest only a modest benefit of PD1/PDL1 inhibitors in this disease. Optimizing immunotherapeutic approaches will require a detailed understanding of the immunogenomic landscape of this disease worldwide. We combined targeted next-generation sequencing and immunohistochemistry to create detailed immunogenomic landscapes from 2 cohorts of gallbladder cancer cases from the United States (n = 60) and Chile (n = 62). Mutations in TP53, SMAD4, KRAS, PIK3CA, ARID2, ARID1A, ATM, FBXW7, ERBB2, and NF1 were found in both the US and Chilean primary cohorts, as well as amplifications in ERBB2, CCNE1, MDM2/CDK4, and CCND1. Despite similar mutation profiles, the immune profiles were distinct, with the Latin American cohort having higher densities of biomarkers associated with CD4+ T cells and PD-1 but lower densities of CD68+ macrophages compared with the North American cohort. Clustering and correlation analyses suggest novel immune subgroups and clinical associations independently of any specific mutations. Additionally, supported by multiplexed single-cell imaging technology, we identified low CD4 and high V-domain Ig suppressor of T cell activation as a candidate biomarker pair of poor outcomes. In summary, our findings highlight the importance of sensitivity to geographic location when considering therapeutic developments and pave a path for further immune investigations of this understudied disease.

BACKGROUND:Risk of recurrence and progression of ductal carcinoma in situ (DCIS) to invasive cancer remains uncertain, emphasizing the need for developing predictive biomarkers of aggressive DCIS. METHODS:Human cell lines and mouse models of disease progression were analyzed for candidate risk predictive biomarkers identified and validated in two independent DCIS cohorts. RESULTS:RNA profiling of normal mammary and DCIS tissues (n = 48) revealed that elevated SOX11 expression correlates with MKI67, EZH2, and DCIS recurrence score. The 21T human cell line model of DCIS progression to invasive cancer and two mouse models developing mammary intraepithelial neoplasia confirmed the findings. AKT activation correlated with chromatin accessibility and EZH2 enrichment upregulating SOX11 expression. AKT and HER2 inhibitors decreased SOX11 expression along with diminished mammosphere formation. SOX11 was upregulated in HER2+ and basal-like subtypes (P < 0.001). Longitudinal DCIS cohort (n = 194) revealed shorter recurrence-free survival in SOX11+ than SOX11- patients (P = 0.0056 in all DCIS; P < 0.0001 in HER2+ subtype) associated with increased risk of ipsilateral breast event/IBE (HR = 1.9, 95%CI = 1.2-2.9; P = 0.003). DISCUSSION:Epigenetic activation of SOX11 drives recurrence of DCIS and progression to invasive cancer, suggesting SOX11 as a predictive biomarker of IBE.

Supplemental Fig. S4. (A) Effect of engineered KRAS expression on response to CDK2 inhibition by seliciclib. KRAS mutation sensitized lung cancer cells towards seliciclib-mediated CDK2 inhibition of growth as compared to control-ED-1 cells. (B) Cells transfected with the plasmid expressing HA-tagged human CP110 were used for detection of CP110 using an anti-CP110 antibody (1:1000) and an anti-HA antibody (1:1000), respectively. The upper band detected by the anti-CP110 antibody specifically recognizes CP110. (C) Respective CP110 and RAS protein expression profile in murine lung cancer cell lines is shown. (D) Effect of KRAS knockdown at 48 hours (left panel) and 72 hours (right panel) on CP110 expression in the 344P cell line. (E) Effect of KRAS knockdown at 72 hours (left panel) and 96 hours (right panel) on CP110 expression in the Hop62 cell line.

Supplemental Table 1. Summary of the clinical and histopathologic characteristics of Merkel cell carcinoma patients. Supplemental Table 2. Summary of antibodies used in this study. Supplemental Table 3. Correlation between tumor associated immune infiltrate and co-localized expression of CD31 and B7-H3 in Primary MCCs.

Median TAIC density by mm2 in the intratumoral and peritumoral compartments of lung ADC and SqCC specimens.

Supplemental File S1. The siRNA sequences and real-time qPCR primer sequences.

Supplemental Figure 3. Correlation between B7-H3/CD31 G-function co-localization index in MCPyV-negative MCCs. Plots showing the correlation B7-H3/CD31 G-function co-localization index for each primary MCPyV-negative MCC plotted against the frequency of associated malignancies (A) and lymphovascular invasion (B). G-function calculated according to a 1 pixel (1.57 ï­m) radius.

Supplemental Table 1. Relationship Between Merkel Cell Polyoma Virus and Clinicopathologic Parameters. Supplemental Table 2. Univariate Logistic Regression Models for Metastasis to Any Site. Supplemental Table 3. Univariate Logistic Regression for Metastasis to Individual Lymph Node Beyond Sentinel Lymph Node. Supplemental Table 4. Univariate Logistic Regression for Metastasis to Sentinel Lymph Node. Supplemental Table 5. Univariate Logistic Regression for Metastasis to Central Nervous System. Supplemental Table 6. Spearman Correlation Between Cell Density and Age, Tumor Size, Depth of Invasion, and Mitotic Figures. Supplemental Table 7. Relationship Between Gender and Immune Infiltrate. Supplemental Table 8. Merkel Cell Polyoma Virus and Immune Infiltrate. Supplemental Table 9. Immune Infiltrate Cell Density: Descriptive Statistics. Supplemental Table 10. Relationship Between Anatomic Site and Immune Infiltrate. Supplemental Table 11. Relationship Between Lymphovascular Invasion (LVI) and Immune Infiltrate. Supplemental Table 12. Relationship Between Perineural Invasion (PNI) and Immune Infiltrate.

<p>Supplemental Fig. S3. (A) Schematic diagram showing the relationship between CDK2, CP110, activated KRAS and anaphase catastrophe. (B) Engineered overexpression of CP110 was detected in A549, Hop62, H522 and H460 cells (versus control transfectants) using anti-HA antibody after 24 and 48 hours of transfection. A representative immunoblot for each cell line is displayed.</p>

Contains supplementary methods and supplementary figure legends.

S1. Introduction of enzymatically-active USP18 overcomes growth inhibition of USP18-repressed lung cancer cells. S2. Loss of USP18 expression reduces growth of KRAS mutant human lung cancer cells. S3. Interferon (IFN) treatment of lung cancer cells having repressed USP18 expression promotes growth inhibition. S4. Modifying USP18 expression alters KRAS protein levels. S5. USP18 regulates KRAS post-transcriptionally. S6. Repression of USP18 expression downregulates AKT and MAPK signaling pathways. S7. KRAS is ISG15-modified. S8. USP18 knockdown promotes KRAS localization to the endomembrane compartment. S9. KrasLA2/+ mice were crossed with Usp18-/- mice to generate KrasLA2/+/Usp18-/- compound mice. S10. KRAS mRNA is increased with presence of activating KRAS mutations and USP18 protein expression is augmented in the presence of high KRAS levels.

Lung cancer (LC) remains as the leading cause of death by cancer worldwide. In Chile, LC is the second cause of cancer-related deaths. Recently, studies in melanoma and non-small cell lung cancer (NSCLC) patients have highlighted the role of the gut microbiota as an important host factor in mediating the responses/resistance to immunotherapy, suggesting that bacteria present in the gut may modulate the immune response in these tumors. However, the role of extra-intestinal microbiota; bacteria living outside of the gut, in cancer pathogenesis and the response to anti-cancer therapies remains largely undetermined. Here we characterize the composition of intratumoral microbiota of NSCLC and seek to establish a functional relationship between it and the composition of the immune microenvironment and the clinicopathological characteristics of NSCLC patients. From the first 157 FFPE NSCLC samples, we extracted DNA from all collected samples, obtaining adequate material in quality and quantity to later be submitted for analysis to 16S sequencing. Using 16S rRNA gene sequencing, we assessed the general landscape of the NSCLC tumor microbiome, revealing the presence of large number of bacterial communities in the NSCLC tumor samples on the different histological subtypes analyzed. We have detected differences in alpha diversity in the histological subtypes studied. Even more interesting, we have detected significant differences in lung adenocarcinoma depending on the degree of histological differentiation, observing a decrease in proteobacteria and an enrichment of Bacteroidales, as cell differentiation is lost. Additionally, we identified taxonomic differences between differentiated and undifferentiated tumors. Differentiated tumors show enrichment in Akkermansia muciniphila and while undifferentiated tumors show enrichment in Actinobacter Corynebacterium. Suggesting that these taxa could contribute to maintaining a differentiated state or inducing cell dedifferentiation, respectively. Our results reveal the presence of a large number of bacterial communities in lung cancer samples in the different histological subtypes analyzed. Suggesting that these communities could play a key role in tumor differentiation and progression. Citation Format: Erick M. Riquelme, Ivania Valdes, Carlos Aravena, Ilse Valencia, Barbara Mino, Daniel Carvajal, Alberto Martin. Revealing the role of lung cancer microbiota in the tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 651.

Correlation between immunohistochemical PD-L1 H-score expression in MCs and TAIC density by mm2 in the intratumoral and peritumoral compartments in lung ADC and SqCC specimens.

Figure S1: The structure of CFI-402257. Figure S2: TTK mRNA expression profiles in lung cancer cases with different smoking patterns, as shown in panel A. In this left panel N means no smoking history while Y indicates the presence of a smoking history. That was displayed in the right panel as never, former or current smokers. Panel B displays the anti-proliferative effects of CFI-402257 relative to vehicle treatments in lung adenocarcinoma cell lines. The area under the curve was calculated based on the proportion of control values and log10 dosage values to generate dose-response curves. Error bars are standard deviations. Figure S3: Immunoblot analysis of ED1 (A) and A549 (B) lung cancer cells after independent transfection with TTK-targeting (siRNA1 and siRNA2) or control siRNAs. Figure S4: Representative photomicrographs of 393P (A) or H1299 (B) lung cancer cell line-derived tumors after 21 days of CFI-402257 or vehicle treatments. (C) Syngeneic 393P tumor-bearing mice were treated with vehicle, 8.5 mg/kg CFI-402257 orally for 5 days, after which tumors were harvested for RPPA analyses (n = 5 mice per group). Comparison of the 8.5mg/kg CFI-402257-treated group to vehicle-treated groups. The proteins displayed above the red line are significantly different (P < 0.05). Figure S5: (A) The association between survival and expression of MAP2K1/MAP2K2 and TTK in combination using TCGA. Green: MAP2K low/TTK low, purple: MAP2K low/TTK high, orange: MAP2K high/TTK low and red: MAP2K high/TTK high. (B) The combination index (CI) for each dosage pair was calculated. The dash line indicates CI=1.0. Figure S6: TTK mutations in lung adenocarcinomas (A) and squamous cell carcinomas (B) in TCGA database.

Supplemental File S2. A representative video of a Hop62 cell that undergoes multipolar cell division.

<p>Supplemental Fig. S1. Effect of wild-type or a phosphorylation sites mutant CP110 species on responses to CDK2 inhibition. (A) Overexpression of CP110 was detected in ED-1 cells with an anti-HA antibody 24 and 48 hours after transfection. (B) Overexpression of wild-type CP110 significantly reduced apoptosis induced by seliciclib treatment. ED-1 cells overexpressing CP110 were treated with the indicated dosages of seliciclib for 24 hours and analyzed for apoptosis. (C and D). Overexpression of phosphorylation-site mutated CP110 did not have a significant effect on anaphase catastrophe or apoptosis induced by CDK2 inhibition. ED-1 cells overexpressing a mutant-CP110 species were treated with the indicated dosages of seliciclib for 24 hours or transfected individually with two different siRNAs targeting CDK2 for 24 hours and (C) scored for multipolar anaphase and (D) analyzed for apoptosis. (E). Seliciclib treatment did not appreciably affect CP110 basal levels in human and murine lung cancer cells. Hop62 and ED-1 were treated with various dosages of seliclcib and CP110 levels were examined after 24 and 48 hours of treatment.</p>

<p>Supplemental Fig. S2. Overexpression of wild-type CP110, but not phosphorylation-sites mutated CP110 in LKR13 murine lung cancer cells that are driven by KRAS rescued anaphase catastrophe and reduced apoptosis induced by pharmacological CDK2 inhibition after seliciclib treatment. LKR13 cells overexpressing wild-type CP110 species or an empty vector, a phosphorylation-sites mutant-CP110 species (MUT-CP110) or an empty vector (Vector) were treated with indicated dosages of seliciclib. After 24 hours treatment, LKR13 cells were (A) scored for multipolar anaphase and (B) analyzed for apoptosis, as detected by Annexin V:FITC and 7-aminoactinomycin D (7-AAD) staining.</p>