{'fraction_non_alphanumeric': 0.03260869565217391, 'fraction_numerical': 0.018506545983513818, 'mean_word_length': 5.300661914460285, 'pattern_counts': {'":': 0, '<': 2, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
The role of SOX9 in non-small cell lung cancer progression is histopathology-selective

The transcription factor SOX9 is a key regulator of multiple developmental processes, and is frequently re-expressed in non-small cell lung cancer (NSCLC). Its precise role in the progression of NSCLC histopathologies has however remained elusive. We show that SOX9 expression relates to poor outcome and invasive histopathology in human adenocarcinomas, and is absent in murine early minimally invasive and human in situ adenocarcinoma. Interestingly, despite wide SOX9 expression across advanced NSCLC histotypes, its genetic deletion in the murine KrasG12D;Lkb1-/- model selectively disrupted only the growth of papillary NSCLC, without affecting the initiation of precursor lesions or growth of mucinous or squamous tissue. Spatial tissue phenotyping indicated a requirement of SOX9 expression for the progression of surfactant protein C-expressing progenitor cells, which gave rise to papillary tumours. Intriguingly, while SOX9 expression was dispensable for squamous tissue formation, its loss in fact led to enhanced squamous tumour metastasis, which was associated with altered collagen IV deposition in the basement membrane. Our work therefore demonstrates histopathology-selective roles for SOX9 in NSCLC progression, namely a requirement for papillary adenocarcinoma progression, but opposing metastasis-suppressing function in squamous histotype tissue. This attests to a pleiotropic SOX9 function, linked to the cell of origin and microenvironmental tissue contexts.


Introduction
Lung cancer remains the leading cause of cancer-related mortality, the majority of which represent non-small cell lung cancer (NSCLC; 85%). NSCLC is subdivided via its two major histotypes, adenocarcinoma (AC) and squamous cell carcinoma (SCC), of which the AC group is most heterogeneous [1][2][3][4]. ACs are further subdivided based on the tissue's predominant histotype structure, particularly papillary AC (PAC), mucinous AC (MAC), and acinar AC (AAC) [5]. Certain driver mutations are selectively enriched in these AC subtypes. For example, KEAP1 mutation or NRF2 overexpression occurs more frequently in PACs when compared to total NSCLCs, and KRAS, EGFR or ALK mutations have respectively been associated with mucinous, lepidic growth, or signet ring structure [6,7]. Molecular and genetic functions therefore need to be interpreted within these histopathology-specific tissue contexts.
The transcription factor SOX9 is a member of the SOX (sex-determining region Y-related) family [8], and regulates a variety of developmental processes [9][10][11]. During murine lung development, SOX9 is expressed in a timed manner to coordinate lung branching morphogenesis and alveolar differentiation [12,13]. SOX9 is silenced in adult tissues but frequently re-expressed in cancer, where it has been associated with increased cell proliferation, transcriptional reprogramming and self-renewal of stem cells, and increased cell invasiveness and drug resistance [14][15][16][17]. The SOX9 gene resides on the long arm of chromosome 17 (17q24.3), which is frequently gained in non-smokers with lung AC [18]. In patients, elevated SOX9 expression has been associated with poor survival and advanced clinical NSCLC stage, and its expression was shown to promote the proliferation of AC cells in vitro through affecting the CDKN1A and CDK4 cell cycle regulators [19,20]. However, while many studies find oncogenic roles for SOX9, supporting its therapeutic targeting, also the downregulation of SOX9 has been linked to relapse of prostate cancer [21] and stage II colon cancer [22], indicating that SOX9 has pleiotropic functions. Despite NSCLC constituting a heterogeneous Histopathology-selective roles of SOX9 in lung cancer progression 4 set of diseases, the potential histopathology-selective role of SOX9 in NSCLC progression has thus far not been studied.
Distinct sets of developmental signals and transcription factors are expressed in different lung progenitor cells, and thereby exert lineage-specific functions [23]. In recent years, the ways in which different cells of origin influence NSCLC progression have been addressed by applying genetically engineered mouse models [24]. Using adenovirus-mediated Cre-recombination, we previously showed that conditional Kras G12D   Clinicopathological and follow-up data was obtained from regional and national databases, and all morphological assessments were performed by a subspecialty consultant diagnostic histopathologist.
Histopathology-selective roles of SOX9 in lung cancer progression

Tissue processing, immunohistochemistry (IHC) and image acquisition
Murine lung tumours were processed and embedded in paraffin as previously described [23].
FFPE tissue blocks containing whole murine lung or TMA blocks were cut as 4 µm thick sections. Two sequential sections were placed on one glass slide (SUPERFROST ® PLUS, Thermo scientific, Ref J1800AMNZ), and labelled to mark the sectioning order. Adjacent tissue sections were each stained with one antibody. Standard IHC was performed with the primary antibodies listed in supplementary material, Table S1. For routine signal development, BrightVision poly-HRP goat anti-rabbit IgG (Immunologic B.V., no dilution) was incubated at room temperature for 30 min. For developing the SOX2 signal, DaG-HRP (Santa Cruz Biotechnology sc2020, 1:100) was incubated at room temperature for 30 min. For signal detection, Bright-DAB Substrate Kit (Immunologic B.V.) was used. Whole slide bright-field scans of stained murine and clinical tissue sections were acquired by a digital slide scanner (Pannoramic 250, 3DHISTECH Ltd) using a 20x objective. 3 µm sections from the TMA-2 human adenocarcinoma cohort were duplex stained using a Ventana autostainer with antibodies for cytokeratin AE1/AE3 (1:250, #NCL-L-AE1/AE3, Leica detected with Roche discovery yellow chromogen) and SOX9 (1:10000, AP5535, Millipore detected with Roche discovery purple chromogen). Up to three 1 mm cores per tumour were analysed. Images of haematoxylin-counterstained images were acquired using a Hamamatsu nanozoomer.

Region-of-interest (ROI)-specific image analysis
Histopathology classification was performed in conjunction with an expert pathologist as previously described [23,24]. The sizes of individual tumours and lung tissues were measured using Pannoramic CaseViewer (3DHISTECH Ltd). For image analysis, TIFF images of murine lungs with hematoxylin and eosin (H&E) and biomarker stainings on whole slide scans were exported using the Pannoramic CaseViewer (3DHISTECH Ltd) at a magnification of 1:2.

Image analysis of human adenocarcinoma cohort (TMA-2)
SOX9 duplex core images were quantified with a purpose-made Visiopharm app. In brief, images were segmented into epithelial and stromal elements using the yellow channel with manual assistance where necessary. Tumour cell nuclei were categorised as weak, negative, moderate or strong for purple SOX9 staining using human-defined cut-offs, and an H-score was calculated for each core (%weak + 2x%moderate + 3x%strong nuclei, yielding a score from 0-300). Ki-67 staining was performed with the Ventana pre-diluted ready-to-use kit, and assessed by a trained histopathologist.
Histopathology-selective roles of SOX9 in lung cancer progression 8

Statistical analysis
For clinical sample (TMA-1) and murine sample analysis, data visualisation and statistical analyses were performed with GraphPad Prism 8 (GraphPad Software, Inc. San Diego, USA).
For statistical comparisons, one-way ANOVA multiple comparison (Kruskal-Wallis test, nonparametric test), unpaired t test (non-parametric Mann-Whitney test), or Fisher's exact test was used. Statistical significance of survival was assessed with a Log-rank (Mantel-Cox) test. P values < 0.05 were considered significant. For the TMA-2 human adenocarcinoma cohort all statistics were performed in R. The cut-off for binarisation of SOX9 was obtained by a published method that has a greater power and lower bias than logrank optimisation in large data sets [31]. The proportional hazards assumption was tested by examination of log-log plots.

The correlation between SOX9 expression and NSCLC staging is histopathology-selective
To evaluate SOX9 expression in NSCLC histotypes according to their stages, we performed IHC analyses of TMAs encompassing 28 human lung SCCs, 13 human lung ASCs and 25 human lung PACs. SOX9 expression was not seen in normal lung tissues but observed in the tumour regions in most of all samples (SCC: 96%, ASC: 100%, PAC: 88%). This significantly contrasts with the expression of SOX2, a well-established genetic driver of SCC, which was more commonly detected in SCCs and ASCs than PACs (SCC: 85%, ASC: 100%, PAC: 8%) ( Figure 1A). The SOX9 protein was mainly located in the nucleoplasm, but cytoplasmic/membranous expression was also observed, particularly in PACs ( Figure 1A-B).
Based on visual scoring, we assigned each sample to a 'SOX9-negative', 'SOX9-moderate' or 'SOX9-high' category and grouped them by their clinical stages ( Figure 1B-C). Among the three NSCLC histotypes, PACs contained the most SOX9-negative or -moderate cases, particularly in the lowest stage tumours (6/6 cases in stage I and 5/7 cases in stage II), whereas in SCCs and ASCs absence of SOX9 was only detected in one sample and at least half of all stage I-II cases were SOX9-high. Thus, a positive correlation between SOX9 expression and clinical stage was detected in PACs, but not in ASCs or SCCs.
To more deeply explore the relationship between SOX9 and the phenotype of human lung adenocarcinoma, we quantified the expression of SOX9 expression in epithelial tumour cell nuclei in a large cohort of 970 resected primary lung adenocarcinomas in tissue microarrays ( Figure 1D). Interestingly, nuclear SOX9 levels were higher in mucinous than non-mucinous tumours (p<0.001), and cores with invasive growth patterns showed a non-significant trend towards higher levels of SOX9 expression selectively in non-mucinous samples (p=0.09 in non-mucinous; p=0.58 in mucinous) (Fig 1D-E). Finally, corroborating previous studies [19-Histopathology-selective roles of SOX9 in lung cancer progression 10 20, 32], epithelial tumour cell proliferation assessed by the percentage of tumour nuclei expressing Ki-67 was significantly related to SOX9 expression (p=0.003) (Fig 1E), and in analysis of patient survival time after surgery higher median levels of SOX9 related to relatively poor outcome (Cox model HR=1.2, p=0.044) (supplementary material, Figure S1).

SOX9 is commonly expressed in advanced murine Kras G12D ;Lkb1 -/tumours
We subsequently applied animal models to study the role of SOX9 in NSCLC histotype formation. As a first step, we examined SOX9 expression in tumours from Kras G12D ;Lkb1 -/mice (KL mice characterised in [25], in this study named KLS wt ). Most KLS wt histotypes, including ASC, MAC and PAC, displayed high SOX9 expression, but its expression was fully absent or low in IACs which are small and represent the least aggressive lesion type with low proliferation and oncogenic signalling activities ( Figure 2; supplementary material, Figure   S2A). Therefore, SOX9 expression levels in murine NSCLC histotypes were comparable to those in clinical NSCLC, showing absent or low expression in IAC or in situ non-mucinous adenocarcinomas and wide expression across all advanced NSCLC histotypes.
Kaplan-Meier survival analysis showed significantly longer survival of KLS het -SPC and KLS null -SPC mice, as well as KLS null -CC10 mice, compared to the relative KLS wt controls ( Figure 3B). The increased life span of KLS het -SPC and KLS null -SPC cohorts corresponded to a reduced PAC burden ( Figure 3C), and no papillary tissue structure was observed in SOX9negative tumours in these mice ( Figure 3C; supplementary material, Figure S2C). In Ad5-CC10-Cre cohorts, Sox9 loss was associated with a shift in the major tumour burden from ASC to SCC tissue with large necrotic regions ( Figure 3D). These SCC tumours were positive for the AC marker NKX2-1 and lacked SOX2 expression (supplementary material, Figure S2B), suggesting that these were derived from ASCs, and hence called SCC-like from here onwards.
Interestingly, only the formation of ASC with papillary AC cores, but not those with mucinous AC cores, was disrupted ( Figure 3D and supplementary material, Figure S2C). Additionally, PAC was the only AC subtype normally present in KLS wt -CC10 mice that was absent following Sox9 loss ( Figure 3D and supplementary material, Figure S2C).
Together, this data suggested that SOX9 expression was selectively required for the growth of papillary histotype tissue. In support of this conclusion, SOX9-positive tumours in both the Sox9 null -SPC and -CC10 cohorts, explained by recombination escape known to arise in threeallelic compound mice [33], always showed a papillary tissue structure (supplementary material, Figure S3). As expected, based on the obligate role of LKB1 as a tumour suppressor in this model, we found no evidence for escape of Lkb1 recombination (IHC staining; data not shown). Importantly, in both the CC10-and SPC-infected cohorts, KLS het resembled KLS null mice in terms of survival, histopathology spectra and tumour burden (Figure 3B-D; supplementary material, Figure S3C-D), together indicating that both heterozygous and homozygous Sox9 loss selectively disrupted papillary NSCLC histotype formation.

SOX9 expression is essential for the oncogenic transformation of cancer-initiating SPC+ cells
To understand why SOX9 selectively regulated the formation of papillary tissue structure, we studied the spatial expression of cell lineage markers and SOX9 in different tumour histotypes.
In the Ad5-SPC-Cre cohorts, SPC, which marks AT2 progenitor cells and differentiated AT2 cells, was highly expressed in IACs, while SPC was absent or expressed at low levels in PACs supplementary material, Figure S4A), indicating that also CC10-Cre induced the transformation of SPC+ cells. Other tumour histotypes found in Ad5-CC10-Cre cohorts that were not affected by Sox9 loss ( Figure 3D), particularly mucinous and squamous tissue, were always SPC-negative ( Figure 4D and supplementary material, Figure S4A). Interestingly, we further found that SPC expression was mutually exclusive with SOX9 expression in PACs as well as papillary AC cores of larger ASC tumours, and marked tumour subregions that lacked prominent papillary structure ( Figure 4B and D). The concomitant appearance of papillary structure together with SPC loss upon SOX9 expression, together with the absence of PAC and papillary AC cores following Sox9 loss as shown in the section above, suggested that SOX9 was a driving force for papillary tumour progression from SPC+ progenitor cells (illustrated in Figure 4E).

SOX9 expression differently regulates AC and SCC tissue metastases
Given the previous links between SOX9 expression and invasion, we next asked how Sox9 loss affected the metastatic potential of NSCLC histotypes by analysing the mediastinal lymph nodes (supplementary material, Figure S4B-C). Metastases in KLS wt -SPC (observed in 3/8 KLS wt -SPC mice) were similar to those previously described [32] and represented AC histotype tissue with papillary structure (supplementary material, Figure S4C). Metastases that eventually developed in KLS null -SPC mice (4/19 mice, including 2 minimal metastases) were always SOX9-positive, suggesting that they had originated from PACs that had escaped Sox9 recombination ( Figure 5A and C). Also, the KLS het -SPC mice developed frequent metastasis (12/17 mice, including 6 minimal metastasis), which could however not be linked to SOX9 status due to the heterozygous SOX9 expression. Importantly, as previously reported [32], squamous metastases were never detected in KLS wt -CC10 mice, but observed in 2/11 KLS het -CC10 and 2/13 KLS null -CC10 mice ( Figure 5B and D; supplementary material, Figure S4D).
Three of the four mice carrying squamous metastases succumbed to their disease relatively early and within the survival range of control KLS wt mice. This analysis therefore suggested an overall reduction in AC metastases following Sox9 loss, linked to a block in PAC progression, mirrored by a notable increase in SCC-like metastases following Sox9 loss in murine NSCLC.
As SOX9 is known to regulate extracellular matrix (ECM) deposition, we next examined ECM features following Sox9 loss by staining of collagen IV (COLIV), a central component of the tumour basement membrane. COLIV was expressed in both IAC and PAC, but only in the latter it formed a linear structure, which was also observed in papillary SOX9-expressing tissue at metastatic sites ( Figure 6A-B). This suggested that a precise alignment of COLIV, regulated by SOX9 expression, might be required for the actual growth and later invasiveness of PACs.
On the other hand, in Sox9 wt ASCs, COLIV formed continuous boundaries around squamous tumour cell pockets ( Figure 6C), while Sox9 null SCC-like tumours and the SCC regions of ASCs with mucinous AC cores showed thinner parallel COLIV fibres as well as granular and focal depositions ( Figure 6D). A similar alteration in COLIV rearrangement was also observed in the SCC-like LN metastases ( Figure 6D). Taken together, in murine PACs, Sox9 loss reduced primary papillary tumour progression and consequently their invasion, possibly related to an alteration in ECM deposition, while in squamous histotype the altered COLIV deposition following Sox9 loss in fact associated with an increase in tumour invasiveness and metastasis (illustrated in Figure 6E).

Discussion
Re-expression of the lung developmental transcription factor SOX9 specifically in malignant lung tissues, in conjunction with the reported positive correlation of SOX9 expression levels with clinical NSCLC staging, renders SOX9 a good candidate for prognostic and therapeutic interrogation. We here studied the previously unexplored expression and role of SOX9 in NSCLC histotypes. We identified a trend towards elevated SOX9 expression in non-mucinous clinical invasive AC compared to in situ samples, as well as in murine PAC compared to IAC, with IAC constituting an early form of PAC. SOX9 was widely expressed across multiple invasive stage murine and clinical NSCLC histotypes, yet murine Sox9 loss only affected papillary structure formation, not the initiation or progression of other histotype lesions. The overarching conclusion of our work is therefore that the role of SOX9 in NSCLC is to be interpreted in a histotype-selective manner.
The expression of Kras G12D combined with Lkb1 loss in alveolar AT2 or bronchial Clara lung progenitor cells gives rise to a range of NSCLC histotype tumours. While the alveolar differentiation marker SPC was uniformly expressed in SOX9-IAC, its downregulation coincided with the appearance of papillary tissue structure, and SPC was completely absent in mature SOX9-expressing papillary tumour regions. A transition from alveolar dedifferentiation to gradual SOX9 expression was similarly seen following oncogenic KRAS expression in organoids [34], and corroborates data showing that ectopic expression of SOX9 in embryonic lungs prevented AT2 cell differentiation [12,13], overall indicating that SOX9 expression intrinsically associates with AT2 lineage exit. Use of a conditional Sox9 null model to address the role of SOX9 in NSCLC progression showed that Sox9 loss disrupted the formation of PAC and papillary AC cores of ASC lesions, but not IAC or histotypes derived from bronchial progenitors. This therefore suggests that SOX9 expression is specifically required to drive the progression of lesions developed from a SPC+ cell of origin, leading to papillary NSCLC formation. Precisely how SOX9 drives AT2 lineage exit remains unclear, and an intriguing future direction would hence be to identify the lineage-specific SOX9 transcriptional network, and particularly its interplay with transcription factors known to regulate alveolar differentiation, such as FOXA2 [35-37]. Our study thus reveals a role of SOX9 in NSCLC progression that is selectively linked to the SPC+/AT2 lineage of cancerinitiating lung progenitor cells.
We showed that Sox9 loss decreased AC metastasis, which is consistent with a reported prometastatic role of SOX9 in multiple cancer types [38][39][40]. But surprisingly, the incidence of SCC metastasis increased following Sox9 loss. One mechanism in which SOX9 is involved in tumour metastasis is through regulating the expression and deposition of ECM components, including multiple collagens and laminins [14]. Our findings importantly suggest that SOX9regulated ECM re-arrangements affect tumour invasiveness in different and microenvironmental context-dependent manners. We identified differential COLIV deposition, which marks tumour basement membranes, across NSCLC histotypes. Linear COLIV was observed in SOX9-expressing primary PACs and papillary metastatic LNs, but not in nonmetastatic SOX9-negative IACs, suggesting that it is part of a supportive structure for papillary growth and metastasis upon SOX9 expression. On the other hand, in the SOX9-expressing squamous regions COLIV formed pocket structures surrounding the tumour islets, and these became discontinuous and granular in SOX-negative squamous region. Similar COLIV discontinuity has been previously linked to the progression of oral SCC [41], suggesting that disrupted squamous pocket structure is commonly associated with increased squamous invasiveness. Our study therefore depicts functional heterogeneity of SOX9 in NSCLC Histopathology-selective roles of SOX9 in lung cancer progression metastasis, namely a requirement for SOX9-associated matrix deposition in papillary histotype metastasis, but an opposing metastasis-suppressing function in squamous histotype tissue.
Analysis of Sox9 heterozygous null mice showed that loss of one Sox9 allele already disrupted papillary tissue formation. While Sox9 haploinsufficiency has not been previously described in cancer studies, loss of one Sox9 allele has been associated with defects in beta-cell development A similar strategy could possibly be applied to identify small molecules that disrupt SOX9 Histopathology-selective roles of SOX9 in lung cancer progression 18 dimerisation or its binding to other lineage-specific transcription factors. However, while our study implies SOX9 as a potential anti-cancer target in papillary histotype NSCLC, Sox9 loss also associated with enhanced squamous tissue metastasis, emphasising that therapeutic considerations ought to consider the tumour's histotype context and cannot simply adopt SOX9 positivity as a biomarker for response.
In conclusion, we demonstrate opposing requirements for SOX9 in different NSCLC histotypes, and shed light on a pleiotropic SOX9 function that links to different tumour progenitor cells and extracellular microenvironmental features. This provides a strong motivation to evaluate the prognostic value and therapeutic application of SOX9 in the context of NSCLC histopathology types.

Acknowledgements
We thank the Digital and Molecular Pathology Unit supported by the University of Helsinki and Biocenter Finland, and the Laboratory Animal Centre for husbandry support. We thank Zhangyi He for performing statistical analysis on the TMA-2 dataset. Katja Välimäki is thanked for material support, Ashwini Nagaraj for advice, and Iris Lähdeniemi for reviewing the manuscript. Samples/data used for the research were obtained from the Helsinki Biobank, and we warmly thank all patients for consenting to archive their samples in the Biobank.

Data availability statement
The data that support the findings of this study, including murine tumour and TMA-1 analyses, are available as supplementary materials, Table S2       These IAC lesions gradually start to express SOX9 and this is essential for papillary tumour progression; the SPC+ identity is gradually lost when papillary tissue structure appears. Upon Ad5-CC10-cre infection, SPC+ IACs again develop, either from AT2 cells that are targeted by the CC10-cre virus or from trans-differentiated Clara or dual positive SPC+/CC10+ cells.
SOX9 expression in papillary tissue corresponds with loss of SPC expression, giving rise to PACs or the papillary AC core of ASC. Lastly, transformation of CC10+ progenitor cells, which are mostly Clara cells, gives rise to MAC, AAC and ASC with mucinous AC core. Right: Sox9 loss selectively disrupts papillary histotype tissue formation in which transformed cells exhibit progression from a SPC+ to a SPC-status.