Patient-derived non-small cell lung cancer xenograft mirrors complex tumor heterogeneity

Objective:Patient-derived xenograft(PDX)models have shown great promise in preclinical and translational applications,but their consistency with primary tumors in phenotypic,genetic,and pharmacodynamic heterogeneity has not been well-studied.This study aimed to establish a PDX repository for non-small cell lung cancer(NSCLC)and to further elucidate whether it could preserve the heterogeneity within and between tumors in patients.Methods:A total of 75 surgically resected NSCLC specimens were implanted into immunodeficient NOD/SCID mice.Based on the successful establishment of the NSCLC PDX model,we compared the expressions of vimentin,Ki67,EGFR,and PD-L1 proteins between cancer tissues and PDX models using hematoxylin and eosin staining and immunohistochemical staining.In addition,we detected whole gene expression profiling between primary tumors and PDX generations.We also performed whole exome sequencing(WES)analysis in 17 first generation xenografts to further assess whether PDXs retained the patient heterogeneities.Finally,paclitaxel,cisplatin,doxorubicin,atezolizumab,afatininb,and AZD4547 were used to evaluate the responses of PDX models to the standard-of-care agents.Results:A large collection of serially transplantable PDX models for NSCLC were successfully developed.The histology and pathological immunohistochemistry of PDX xenografts were consistent with the patients’tumor samples.WES and RNA-seq further confirmed that PDX accurately replicated the molecular heterogeneities of primary tumors.Similar to clinical patients,PDX models responded differentially to the standard-of-care treatment,including chemo-,targeted-and immuno-therapeutics.Conclusions:Our established PDX models of NSCLC faithfully reproduced the molecular,histopathological,and therapeutic characteristics,as well as the corresponding tumor heterogeneities,which provides a clinically relevant platform for drug screening,biomarker discovery,and translational research.

Intratumor heterogeneity,microenvironment,and mechanisms of drug resistance in glioma recurrence and evolution

Glioma is the most common lethal tumor of the human brain.The median survival of patients with primary World Health Organization grade IV glioma is only 14.6 months.The World Health Organization classification of tumors of the central nervous system categorized gliomas into lower-grade gliomas and glioblastomas.Unlike primary glioblastoma that usually develop de novo in the elderly,secondary glioblastoma enriched with an isocitrate dehydrogenase mutant typically progresses from lower-grade glioma within 5-10 years from the time of diagnosis.Based on various evolutional trajectories brought on by clonal and subclonal alterations,the evolution patterns of glioma vary according to different theories.Some important features distinguish the normal brain from other tissues,e.g.,the composition of the microenvironment around the tumor cells,the presence of the blood-brain barrier,and others.The underlying mechanism of glioma recurrence and evolution patterns of glioma are different from those of other types of cancer.Several studies correlated tumor recurrence with tumor heterogeneity and the immune microenvironment.However,the detailed reasons for the progression and recurrence of glioma remain controversial.In this review,we introduce the different mechanisms involved in glioma progression,including tumor heterogeneity,the tumor microenvironment and drug resistance,and their pre-clinical implements in clinical trials.This review aimed to provide new insights into further clinical strategies for the treatment of patients with recurrent and secondary glioma.

Single-cell RNA sequencing in breast cancer: Understanding tumor heterogeneity and paving roads to individualized therapy

Single-cell RNA sequencing(scRNA-seq)is a novel technology that allows transcriptomic analyses of individual cells.During the past decade,scRNA-seq sensitivity,accuracy,and efficiency have improved due to innovations including more sensitive,automated,and cost-effective single-cell isolation methods with higher throughput as well as ongoing technological development of scRNA-seq protocols.Among the variety of current approaches with distinct features,researchers can choose the most suitable method to carry out their research.By profiling single cells in a complex population mix,scRNA-seq presents great advantages over traditional sequencing methods in dissecting heterogeneity in cell populations hidden in bulk analysis and exploring rare cell types associated with tumorigenesis and metastasis.scRNA-seq studies in recent years in the field of breast cancer research have clustered breast cancer cell populations with different molecular subtypes to identify distinct populations that may correlate with poor prognosis and drug resistance.The technology has also been used to explain tumor microenvironment heterogeneity by identifying distinct immune cell subsets that may be associated with immunosurveillance and are potential immunotherapy targets.Moreover,scRNA-seq has diverse applications in breast cancer research besides exploring heterogeneity,including the analysis of cell-cell communications,regulatory single-cell states,immune cell distributions,and more.scRNA-seq is also a promising tool that can facilitate individualized therapy due to its ability to define cell subsets with potential treatment targets.Although scRNA-seq studies of therapeutic selection in breast cancer are currently limited,the application of this technology in this field is prospective.Joint efforts and original ideas are needed to better implement scRNA-seq technologies in breast cancer research to pave the way for individualized treatment management.This review provides a brief introduction on the currently available scRNA-seq approaches along with their corresponding strengths and weaknesses and may act as a reference for the selection of suitable methods for research.We also discuss the current applications of scRNA-seq in breast cancer research for tumor heterogeneity analysis,individualized therapy,and the other research directions mentioned above by reviewing corresponding published studies.Finally,we discuss the limitations of current scRNA-seq technologies and technical problems that remain to be overcome.

Intratumoral heterogeneity of hepatocellular carcinoma: From singlecell to population-based studies

Hepatocellular carcinoma(HCC)is characterized by high heterogeneity in both intratumoral and interpatient manners.While interpatient heterogeneity is related to personalized therapy,intratumoral heterogeneity(ITH)largely influences the efficacy of therapies in individuals.ITH contributes to tumor growth,metastasis,recurrence,and drug resistance and consequently limits the prognosis of patients with HCC.There is an urgent need to understand the causes,characteristics,and consequences of tumor heterogeneity in HCC for the purposes of guiding clinical practice and improving survival.Here,we summarize the studies and technologies that describe ITH in HCC to gain insight into the origin and evolutionary process of heterogeneity.In parallel,evidence is collected to delineate the dynamic relationship between ITH and the tumor ecosystem.We suggest that conducting comprehensive studies of ITH using single-cell approaches in temporal and spatial dimensions,combined with population-based clinical trials,will help to clarify the clinical implications of ITH,develop novel intervention strategies,and improve patient prognosis.

Heterogeneity and renal mass biopsy:a review of its role and reliability

Increased abdominal imaging has led to an increase in the detection of the incidental small renal mass(SRM). With increasing recognition that the malignant potential of SRMs is heterogeneous, ranging from benign(15%-20%) to aggressive(20%), enthusiasm for more conservative management strategies in the elderly and infirmed, such as active surveillance(AS), have grown considerably. As the management of the SRM evolves to incorporate ablative techniques and AS for low risk disease, the role of renal mass biopsy(RMB) to help guide individualized therapy is evolving. Historically, the role of RMB was limited to the evaluation of suspected metastatic disease, renal abscess, or lymphoma. However, in the contemporary era, the role of biopsy has grown, most notably to identify patients who harbor benign lesions and for whom treatment, particularly the elderly or frail, may be avoided. When performing a RMB to guide initial clinical decision making for small, localized tumors, the most relevant questions are often relegated to proof of malignancy and documentation(if possible) of grade. However, significant intratumoral heterogeneity has been identified in clear cell renal cell carcinoma(ccRCC) that may lead to an underestimation of the genetic complexity of a tumor when single-biopsy procedures are used. Heterogeneous genomic landscapes and branched parallel evolution of ccRCCs with spatially separated subclones creates an illusion of clonal dominance when assessed by single biopsies and raises important questions regarding how tumors can be optimally sampled and whether future evolutionary tumor branches might be predictable and ultimately targetable. This work raises profound questions concerning the genetic landscape of cancer and how tumor heterogeneity may affect, and possibly confound, targeted diagnostic and therapeutic interventions. In this review, we discuss the current role of RMB, the implications of tumor heterogeneity on diagnostic accuracy, and highlight promising future directions.

Heterogeneity and function of cancer-associated fibroblasts in renal cell carcinoma

With the advancement of anticancer therapy,there is increasing interest in understanding the tumor microenvi-ronment(TME).Cancer-associated fibroblasts(CAFs)play a pivotal role in the TME and have been the focus of much research in recent years.CAFs play an active role in cancer progression through complex interactions with other cells in the TME,releasing regulatory factors,synthesizing and remodeling the extracellular matrix.How-ever,research on the role of CAFs in renal cell carcinoma(RCC)is still in its nascent stages.Here,we describe the origins and subgroups of CAFs,the roles of CAFs in the development and progression of RCC,the impact of CAFs on RCC prognosis,and the potential of CAFs as treatment targets in RCC.By analyzing CAF subsets,biomarkers,and targeted therapies,we present the significance and contribution of CAFs in RCC research.Furthermore,we highlight the distinct contribution of CAFs in advanced RCC through horizontal comparison with other cancers.This paper provides a comprehensive perspective of recent and foundational studies on the role of CAFs in RCC and other types of cancers and new insights for further study of CAFs in RCC.

Clinical implications of single cell sequencing for bladder cancer

Bladder cancer(BC)is the 10th most common cancer worldwide,with about 0.5 million reported new cases and about 0.2 million deaths per year.In this scoping review,we summarize the current evidence regarding the clinical implications of single-cell sequencing for bladder cancer based on PRISMA guidelines.We searched PubMed,CENTRAL,Embase,and supplemented with manual searches through the Scopus,and Web of Science for published studies until February 2023.We included original studies that used at least one single-cell technology to study bladder cancer.Forty-one publications were included in the review.Twenty-nine studies showed that this technology can identify cell subtypes in the tumor microenvironment that may predict prognosis or response to immune checkpoint inhibition therapy.Two studies were able to diagnose BC by identifying neoplastic cells through single-cell sequencing urine samples.The remaining studies were mainly a preclinical exploration of tumor microenvironment at single cell level.Single-cell sequencing technology can discriminate heterogeneity in bladder tumor cells and determine the key molecular properties that can lead to the discovery of novel perspectives on cancer management.This nascent tool can advance the early diagnosis,prognosis judgment,and targeted therapy of bladder cancer.

Genetics and genomics of prostate cancer

Prostate cancer （PCa） is one of the most common malignancies in the world with over 890 000 cases and over 258 000 deaths worldwide each year. Nearly all mortalities from PCa are due to metastatic disease, typically through tumors that evolve to be hormone-refractory or castrate-resistant. Despite intensive epidemiological study, there are few known environmental risk factors, and age and family history are the major determinants. However, there is extreme heterogeneity in PCa incidence worldwide, suggesting that major determining factors have not been described. Genome-wide association studies have been performed and a considerable number of significant, but low-risk loci have been identified. In addition, several groups have analyzed PCa by determination of genomic copy number, fusion gene generation and targeted resequencing of candidate genes, as well as exome and whole genome sequencing. These initial studies have examined both primary and metastatic tumors as well as murine xenografts and identified somatic alterations in TP53and other potential driver genes, and the disturbance of androgen response and cell cycle pathways. It is hoped that continued characterization of risk factors as well as gene mutation and misregulation in tumors will aid in understanding, diagnosing and better treating PCa.

Advances in novel molecular typing and precise treatment strategies for small cell lung cancer

Small cell lung cancer(SCLC)is a high-grade neuroendocrine(NE)cancer characterized by high circulating tumor-cell burden and early extensive metastasis.Considering the complexity of SCLC genes and the immune microenvironment,their unique molecular heterogeneity profiles have been continuously explored.The understanding of SCLC subtypes has recently changed from traditional"classical"and"variant"types to"NE"and"non-NE"phenotypes and to the subtypes defined by major transcriptional regulators,which indicates the gradual revelation of high intratumoral heterogeneity and plasticity characteristics of SCLCs.Advances in genomics as well as the development of single-cell sequencing analysis and new preclinical models have helped investigators gain many new insights into SCLCs and the development of targeted therapy and immunotherapy strategies.This article provides an overview of changes in molecular typing,tumor heterogeneity,and plasticity and that of advances in the precise treatment of different subtypes of SCLC.

Natural and artificial small RNAs:a promising avenue of nucleic acid therapeutics for cancer

Since the failure of traditional therapy, gene therapy using functional DNA sequence and small RNA/DNA molecules(oligonucleotide) has become a promising avenue for cancer treatment. The discovery of RNA molecules has impelled researchers to investigate small regulatory RNA from various natural and artificial sources and determine a cogent target for controlling tumor progression. Small regulatory RNAs are used for therapeutic silencing of oncogenes and aberrant DNA repair response genes.Despite their advantages, therapies based on small RNAs exhibit limitations in terms of stability of therapeutic drugs, precisionbased delivery in tissues, precision-based intercellular and intracellular targeting, and tumor heterogeneity-based responses. In this study, we summarize the potential and drawbacks of small RNAs in nucleic acid therapeutics for cancer.

Targeting the mechano-microenvironment and liver cancer stem cells:a promising therapeutic strategy for liver cancer

Over the past 2 decades,cancer stem cells(CSCs)have been identified as the root cause of cancer occurrence,progression,chemoradioresistance,recurrence,and metastasis.Targeting CSCs is a novel therapeutic strategy for cancer management and treatment.Liver cancer(LC)is a malignant disease that can endanger human health.Studies are increasingly suggesting that changes in the liver mechanical microenvironment are a primary driver triggering the occurrence and development of liver cancer.In this review,we summarize current understanding of the roles of the liver mechano-microenvironment and liver cancer stem cells(LCSCs)in liver cancer progression.We also discuss the relationship between the mechanical heterogeneity of liver cancer tissues and LCSC recruitment and metastasis.Finally,we highlight potential mechanosensitive molecules in LCSCs and mechanotherapy in liver cancer.Understanding the roles and regulatory mechanisms of the mechano-microenvironment and LCSCs may provide fundamental insights into liver cancer progression and aid in further development of novel therapeutic strategies.

Transcriptome analysis creates a new era of precision medicine for managing recurrent hepatocellular carcinoma

The high incidence of hepatocellular carcinoma(HCC)recurrence negatively impacts outcomes of patients treated with curative intent despite advances in surgical techniques and other locoregional liver-targeting therapies.Over the past few decades,the emergence of transcriptome analysis tools,including real-time quantitative reverse transcription PCR,microarrays,and RNA sequencing,has not only largely contributed to our knowledge about the pathogenesis of recurrent HCC but also led to the development of outcome prediction models based on differentially expressed gene signatures.In recent years,the single-cell RNA sequencing technique has revolutionized our ability to study the complicated crosstalk between cancer cells and the immune environment,which may benefit further investigations on the role of different immune cells in HCC recurrence and the identification of potential therapeutic targets.In the present article,we summarized the major findings yielded with these transcriptome methods within the framework of a causal model consisting of three domains:primary cancer cells;carcinogenic stimuli;and tumor microenvironment.We provided a comprehensive review of the insights that transcriptome analyses have provided into diagnostics,surveillance,and treatment of HCC recurrence.

Proliferation Characteristics of CD133+ Cell Population in Colorectal Cancer

In this study,CD133+ subpopulations were isolated from 41 primary colorectal cancer tissues,the proliferation and cell cycle distribution of the cells were examined without in vitro expansion,and then compared to those of cell lines.The detection of CD133 in colorectal cancer tissues,isolation of CD133+ and CD133-epithelial subpopulations,Ki-67/DNA multiparameter assay and cell volume analysis were flow cytometrically conducted.The results showed that Ki-67 expression was correlated with CD133 level in primary cancer tissues,while cell cycle G 2 /M phase distribution or clinicopathological characteristics was not.In addition,the CD133+ cells showed larger cell volume and higher Ki-67 expression as compared with CD133-cells.But there was no statistically significant difference in G 2 /M phase distribution between the two subpopulations.Our results demonstrated that the CD133+ subpopulation in colorectal cancer tissue contained more actively cycling and proliferating cells,which was not correlated to clinicopathological factors but might contribute to tumor progression and poor clinical outcome.

Improving cancer therapy by targeting cancer stem cells:Directions, challenges, and clinical results

Cancer stem cells （CSC） are a rare cell population withina tumor characterized by the ability to form tumorsfollowing injection into an immunocompromised host.While the role of CSC has been clearly established inanimal models, evidence of their clinical relevance hasbeen harder to demonstrate. A number of markers,or combination thereof, have been used to detect andmeasure, although non-specifically, CSC in almost allhuman tumors. Several pathways have been identifiedas crucial for, but not necessarily unique to, CSC surviva and proliferation, and novel agents have been designed to target such pathways. A number of such agents have entered early phase development. Further, drugs that have long been marketed for non-oncological indications have been redirected to oncology as they appear to affect one or more of such pathways. This article aims to review the available evidence on the clinical relevance of CSC from a drug development standpoint and the results of early phase clinical trials of agents interfering with the above pathways. It also discusses limitations of current clinical trial design and endpoints to demonstrate anti-CSC activity as well as possible strategies to overcome these limitations.

Single-cell transcriptomics reveals tumor landscape in ovarian carcinosarcoma

Objective:The present study used single-cell RNA sequencing(scRNA-seq)to characterize the cellular composition of ovarian carcinosarcoma(OCS)and identify its molecular characteristics.Methods:scRNA-seq was performed in resected primary OCS for an in-depth analysis of tumor cells and the tumor microenvironment.Immunohistochemistry staining was used for validation.The scRNA-seq data of OCS were compared with those of high-grade serous ovarian carcinoma(HGSOC)tumors and other OCS tumors.Results:Both malignant epithelial and malignant mesenchymal cells were observed in the OCS patient of this study.We identified four epithelial cell subclusters with different biological roles.Among them,epithelial subcluster 4 presented high levels of breast cancer type 1 susceptibility protein homolog(BRCA1)and DNA topoisomerase 2-α(TOP2A)expression and was related to drug resistance and cell cycle.We analyzed the interaction between epithelial and mesenchymal cells and found that fibroblast growth factor(FGF)and pleiotrophin(PTN)signalings were the main pathways contributing to communication between these cells.Moreover,we compared the malignant epithelial and mesenchymal cells of this OCS tumor with our previous published HGSOC scRNA-seq data and OCS data.All the epithelial subclusters in the OCS tumor could be found in the HGSOC samples.Notably,the mesenchymal subcluster C14 exhibited specific expression patterns in the OCS tumor,characterized by elevated expression of cytochrome P450 family 24 subfamily A member 1(CYP24A1),collagen type XXIIIα1 chain(COL23A1),cholecystokinin(CCK),bone morphogenetic protein 7(BMP7),PTN,Wnt inhibitory factor 1(WIF1),and insulin-like growth factor 2(IGF2).Moreover,this subcluster showed distinct characteristics when compared with both another previously published OCS tumor and normal ovarian tissue.Conclusions:This study provides the single-cell transcriptomics signature of human OCS,which constitutes a new resource for elucidating OCS diversity.

Heterogeneity of the tumor immune microenvironment and clinical interventions

The tumor immune microenvironment(TIME)is broadly composed of various immune cells,and its heterogeneity is characterized by both immune cells and stromal cells.During the course of tumor formation and progression and anti-tumor treatment,the composition of the TIME becomes heterogeneous.Such immunological heterogeneity is not only present between populations but also exists on temporal and spatial scales.Owing to the existence of TIME,clinical outcomes can differ when a similar treatment strategy is provided to patients.Therefore,a comprehensive assessment of TIME heterogeneity is essential for developing precise and effective therapies.Facilitated by advanced technologies,it is possible to understand the complexity and diversity of the TIME and its influence on therapy responses.In this review,we discuss the potential reasons for TIME heterogeneity and the current approaches used to explore it.We also summarize clinical intervention strategies based on associated mechanisms or targets to control immunological heterogeneity.

Cerebellar Liponeurocytoma Mimicking Cerebellopontine Angle Epidermoid Cyst:A Case Report

Cerebellar liponeurocytoma (CL) described under many different names, is a rare WHO grade I or II well differentiated neurocytic tumor of the cerebellum with focal lipomatous differentiation. Mainly reported in adulthood, it is fought to be a posterior fossa benign tumor. In this paper, we talk about a 64-year-old woman, following up for Parkinson’s disease presented in our department for 7 months’ history of headache and gait disturbance. MRI showed a right cerebellopontine angle (CPA) heterogeneous unlimited tumor mimicking an epidermoid cyst. She underwent a lateral suboccipital craniectomy procedure that permitted obtaining the whole gross total resection of this tumor. The histopathological diagnosis was a cerebellar liponeurocytoma. She was discharged from hospital the 8th day after surgery and was free of symptoms since 5 years.

Delineating the longitudinal tumor evolution using organoid models

Cancer is an evolutionary process fueled by genetic or epigenetic alterations in the genome.Understanding the evolutionary dynamics that are operative at different stages of tumor progression might inform effective strategies in early detection,diagnosis,and treatment of cancer.However,our understanding on the dynamics of tumor evolution through time is very limited since it is usually impossible to sample patient tumors repeatedly.The recent advances in in vitro 3D organoid culture technologies have opened new avenues for the development of more realistic human cancer models that mimic many in vivo biological characteristics in human tumors.Here,we review recent progresses and challenges in cancer genomic evolution studies and advantages of using tumor organoids to study cancer evolution.We propose to establish an experimental evolution model based on continuous passages of patient-derived organoids and longitudinal sampling to study clonal dynamics and evolutionary patterns over time.Development and integration of population genetic theories and computational models into time-course genomic data in tumor organoids will help to pinpoint the key cellular mechanisms underlying cancer evolutionary dynamics,thus providing novel insights on therapeutic strategies for highly dynamic and heterogeneous tumors.

Tumor organoids to study gastroesophageal cancer: a primer

Gastroesophageal cancers are leading causes of cancer death.Our attempts at adopting molecularly based treatment approaches have been slow and ineffective even though we begin to identify specific targetable gene mutations and pathways.It is dear that we should no longer treat all gastroesophageal cancers as a homogeneous disease,which is what we do when we use nonspecific chemotherapy.However,we currently cannot monitor successful gene/pathway targeting,nor understand how/when tumors develop resistance,nor predict which patients will derive maximal benefit.To improve outcomes,we must precisely detail the heterogeneity of these tumors to then individualize cancer therapy as well as develop novel avenues to study and predict treatment effects in individual patients.To this end,patient-derived organoids,in which tumor cells from individual patients are grown in a Petri dish,are a new versatile system that allows for timely expandability,detailed molecular characterization,and genetic manipulation with the promise of enabling predictive assessment of treatment response.In this review,we will explore the development and basic techniques for organoid generation,and discuss the current and potential future applications of this exciting technology to study the basic science of carcinogenesis and to predict/guide cancer patient care in the clinics.

Tumor cell membrane remodeling with universal ligand for CAR-T cells to inhibit solid tumors

Heterogeneity is a major obstacle to the success of CAR-T therapy in treating solid tumors. The complex tumor micro-environment and varying phenotypes of tumor cells might result in antigen escape, drug resistance, and tumor recurrence. To address this issue, we proposed to use lipid-modified fluorescein isothiocyanate(Lip-FITC) as an artificial ligand to normalize the phenotypes of solid tumor cells. In murine cutaneous melanoma and colon adenocarcinoma that prefer to utilize exogenous long-chain fatty acids, we observed much more uptake of Lip-FITC and significantly increased FITC fluorescence on tumor cell membranes than normal cells. This specific exogenous labeling with FITC enhanced the recognition and selectivity of CART cells in solid tumors, bypassing the limitations derived from antigen expression differences in adoptive cell therapies. Lipid metabolism analysis and in vitro experiments demonstrated the sufficient uptake of long-chain fatty acid(LCFAs)-modified Lip-FITC by solid tumor cells, as well as satisfactory ligand assembly on cell membranes. In solid tumor model, the treatment induced the recognition and initiation of CART cells and successfully suppressed tumor progression through T-cell immunity.