Will individual participant data (IPD) be shared publicly (including data dictionaries)?  

Response - NO

Breast cancer BC has affected millions of women globally and is one of the leading cancer causing deaths Additionally triple negative breast cancer TNBC is the most aggressive rarest 15–20 percent and highly invasive with almost 45 percent of the patients having distant metastasis In general TNBC starts from a benign tumor and later becomes malignant by metastasizing to lymph nodes and distant organs such as bone brain kidney and ovary However diagnosis at the pre malignancy stage increases the chances of successful recovery and has almost minimal chance of reoccurrence Additionally the current therapeutic strategy targets hormone receptors and biological pathway involved proteins however in some cases precision medicine development is required to reduce drug failure and combat therapeutic resistance Given this an in depth BC stage specific protein profiling is necessary to enhance clinical diagnosis and monitor their physiological pathways to intensify prognostic efficiency Over the decades researchers have developed a pile of biomarkers from various biological samples such as tissue blood serum and plasma lymph saliva and urine However very few studies have been performed to study the axillary lymph node LN metastasis of TNBC In reference to one such study Pathania et al 2022 performed iTRAQ proteomics of sentinel LN tissue of BC patients to flag extra cellular matrix ECM protein markers In another study Shin et al 2020 reported TUBB2A from a quantitative mass spectrometry study as a novel biomarker for predicting distant metastatic BC Thus the efficacy of mass spectrometry on developing potential disease markers is well defined In view of this in our first objective we have designed this mass spectrometric study to reveal lymph node sentinel and axillary metastatic markers in TNBC patients for early diagnosis and a robust clinical approach In this study a comparative proteomics study will be performed among healthy breast tissue benign tumor tissue sentinel or axillary LN tissue for a better understanding of differentially expressed proteins in LN metastatic patients and their involvement in different biological pathways for the survival of cancer cells This will help to establish potential markers to design protein specific and biological pathway specific drug development Eventually this will increase the therapeutic efficacy in breast cancer management

On the other hand amongst various reasons for cancer progression and metastasis the tumor microbiome has recently developed an interest for researchers to investigate the host cancer cell microbiome interaction for cancer progression suppression Surprisingly BC has a rich 60 percent and diverse tumor microbiome influencing cancer development progression metastasis and treatment response Additionally studies have shown the involvement of different microbes eg E coli H pylori B fragilis F nucleatum in pancreatic cancer colorectal cancer liver cancer and breast cancer by modulating different physiological pathways such as DNA damage cytotoxicity chronic inflammation cell cycle regulation epithelial to mesenchymal transition EMT and apoptosis Culin et al 2021 Interestingly one study has shown that chemotherapy can shift the breast tumor microbiome and specifically microbes correlate with tumor recurrence Chiba et al 2020 Several bacteria have also been reported to aggravate metastasis eg F nucleatum invasion promotes metastasis by suppressing T cell infiltration Parhi et al 2020 colonization of B fragilis in breast ducts induces metastatic progression Parida et al 2021 Given this on the basis of an urgent need to reveal the involvement of intra tumor microbiota in TNBC progression we have designed the next part of our study ie 2nd objective In this part we will investigate the tumor microbial load and diversity based on culturomics study and the impact of tumor microbiome on TNBC cells through various in vitro functional studies to elucidate the role of tumor microbes in cancer progression This study aims to reveal the tumor microbiome mediated TNBC progression and metastasis for the development of promising clinical treatment.

Aims Objectives

Objective 1 A mass spectrometric study to reveal the lymph node metastatic markers in TNBC patients
Objective 2 To check the tumor microbiome effect on TNBC progression.

Design of the Study

case-control study.

Inclusion Criteria
The study population strictly includes diagnostically confirmed TNBC patients only
For the benign tissue TNBC patients with benign tumors are the study population
For the control healthy breast tissue adjacent to the tumor can be resected from the same patient or as per the convenience of the clinicians
For the lymph node tissue TNBC patients with sentinel or axillary LN positive patients are included
For the lymph node tissue control axillary LN negative tissue can be included.

Exclusion Criteria

For the proteomics and microbial study participants undergoing neoadjuvant chemotherapy will be excluded but a sample size of n equals 5 is welcomed for the comparative study However a complete exclusion of participants undergoing neoadjuvant radiotherapy and antibiotic exposure 3 months will be followed A large number of participants undergoing neoadjuvant chemotherapy cannot be included because it will influence the metastatic patients proteomic pattern neoadjuvant radiotherapy patients are excluded due to loss of proteins due to high radiation and antibiotic exposure patients cannot be included due to risk of tumor microbial scarcity for microbial study.

Study Methodology

Sample collection and storage For Proteomics Study

Tissue samples from each cohort i e cohort 1 healthy samples from adjacent breast tissue 5 cm away from the tumor margins for the control cohort 2 benign tumor tissue of TNBC patients cohort 3 axillary lymph node positive sentinel lymph node in case of sample scarcity from the TNBC patients and cohort 4 axillary lymph node negative for lymph node control sentinel lymph node in case of sample scarcity will be resected
The targeted number of each cohort will be n equals 10 for the discovery phase and n equals 20 for the validation phase except the sample size for cohort 4 is n equals 5
The sample size for the proteomics study is 100 Discovery 35 Validation 65 including all the cohorts.

The sources of the control block are Tissue control Cohort 1 healthy tissue samples from adjacent breast tissue 5 cm away from the tumor margins which is diagnostically confirmed as TNBC negative through IHC for the control
Lymph node control Cohort 2 sentinel or axillary lymph node negative samples for lymph node control diagnostically confirmed through IHC
Each tissue sample will be divided into three categories
One set of tissue samples will be taken for diagnostic confirmation through IHC MRI Mammography and CT scan for breast cancer confirmation and its subtype
The other two sets will be stored for proteomic analysis and microbial analysis respectively
Each sample will be collected during the surgical operation and immediately transferred to sterilized cryomolds embedded in optimal cutting temperature compound OCT compound media snap frozen in liquid nitrogen temporarily and stored at minus 80 degree C until further processing view specimen collection and processing protocol.

Mass spectrometry-based proteomic study

The sample size for the proteomics study is 100 Discovery 35 Validation 65 including all the cohorts
After the safe transfer specimens will be diced and mechanically dissociated with a scalpel and placed in labeled 15 ml Eppendorf tubes containing three stainless steel microbeads
Tubes were then submerged in liquid nitrogen for 60 s and immediately homogenized using a mixer mill Retsch MM400 for 2 to 3 cycles at maximum speed 30 Hz vibrational frequency at 60 s per cycle
Samples will be then placed on ice and a cocktail of RIPA buffer and protease inhibitor will be added to each tube resuspended and placed on a rocker for 30 min on ice at 4 degree C
Samples will be ultrasonicated and centrifuged at 15805 x g for 30 min at 4 degree C
The supernatant will be collected to retrieve a desired 2 mg per ml of protein followed by protein quantification through bicinchoninic acid BCA protein assay kit ThermoFisher Scientific San Jose CA and storage at minus 80 degree C
The samples will be first reduced with tris 2 carboxyethyl phosphine TCEP and cysteines will be alkylated by adding iodoacetamide IAA
Subsequently the samples will be digested overnight at 37 degree C in a mixture of urea and Tris HCl at pH 85 containing trypsin Promega Madison WI at an enzyme substrate ratio of 1 to 50 for 16 hours at 37 degree C
The reaction will be stopped by the addition of 90 percent formic acid to a final concentration of 4 percent. For mass spectrometry MS analysis one microgram of digested peptides will run in technical triplicates and biological replicates followed by characterization peptide spectrum analysis through QTOF mass spectrometer Waters Xevo G2 XS IntelliStartTM technology and database search using MASCOT algorithm within Proteome Discoverer 22 ThermoFisher Scientific against the UniProt Human proteome database to obtain peptide and protein identifications

In the discovery phase through quantitative analysis the proteomic profile of the 4 sample groups i e differential expression upregulation and downregulation in healthy benign and axillary lymph node negative control and lymph node positive metastatic stages will be studied. The differentially expressed peptide sequences will be obtained through in depth targeted proteomics in the validation phase Thus it will be helpful to develop potential markers responsible for TNBC lymph node metastasis and enhance the process of early diagnosis and prognosis.

Tissue Sample Procurement Procedure (For Tumor Microbial Study)

The sample size for the Microbiome study is 65 Cohort 1 n equals 20 Cohort 2 n equals 20 Cohort 3 n equals 20 Cohort 4 n equals 5
For the microbial analysis the samples collected from the cohort as mentioned earlier will be placed immediately in sterile study code labeled tubes suspended in a cocktail mixture of Glycerin DMSO supplemented with fetal bovine serum FBS kept at minus 20 degree C slow freezing and then transferred to minus 80 degree C freezer until processing
An environmental control open tube containing PBS will be placed during the surgery at the operation theatre
The tissue adjacent to the collected breast tissue sample will be evaluated for no tumor or lesion present and histologically normal.

Tumor microbial culture

To isolate anaerobic or aerobic bacteria tissue pieces around 025g will be homogenized in 1 mL chilled PBS under aseptic conditions
For aerobic culture 100uL sample homogenate will be plated on Columbia blood agar CBA with 5 percent sheep blood Man Rogosa Sharpe Medium M0303 and BHI brain heart infusion at 37 degree C aerobically with 5 percent CO2
Subsequently 100uL sample homogenate will be plated on Schaedler anaerobe agar for anaerobic culture in an anaerobic chamber hypoxystation
The plates will be incubated at 37 degree C for either 3 days in aerobic conditions or for 5 days in anaerobic conditions
For the identification of bacteria strains colonies will be picked and streaked in designated plates and conditioned for 1 to 3 days to get single colonies
The single colony was picked to grow in a liquid medium for example MRS media and the Ultra Deep Microbiome Prep kit Molzym Bremen Germany will be used to isolate microbial genomic DNA gDNA from living microbes according to the manufacturers protocol
gDNA drawn from each clinical sample was amplified using an Ion 16S Metagenomic Kit Life Technologies Carlsbad CA USA
Using this kit seven of nine hypervariable regions of the 16S rRNA gene will be amplified through two sets of primers one set for the V2 V4 and V8 regions and the other for the V3 V6 plus V7 and V9 regions

The PCR will be performed by preparing two reactions for each clinical sample for each of the two primer sets using a PCR Sprint thermocycler ThermoHybaid Ashford UK following the kit manufacturers instructions
For the identification and classification of the specific abundant bacterial colonies PCR products will be sequenced with an Ion PGM Sequencer using an Ion PGM HiQ
BAM files obtained from Ion PGM Sequencer output will be directly processed with Ion Reporter Software 56 Life Technologies Carlsbad CA USA
Within this programme the 16S Metagenomic workflow works with MicroSEQ 16S Reference Library v20131 and Greengenes v135 databases to align and obtain a taxonomic identification of sequences
Raw data will first be analysed with Ion Reporter Software 56 Life Technologies Carlsbad CA USA to obtain information on relative taxonomic abundances from primers
Thus the identification and classification of the microbial species will be performed.

Cell culture-based in vitro assays

After culturomics based microbial identification and extraction with the help of different bioinformatics and statistical tools e g Ion Reporter Software 56 Nearest Alignment Space Termination45 NAST algorithm the microbial diversity and abundance of different microbes in healthy benign and lymph node metastatic samples can be estimated
Later for the in vitro studies MDA MB 231 TNBC cell line ATCC will be co cultured with the identified most abundant and diversely found tumor bacteria to study the effect of tumor associated bacteria on cancer cells growth
The isolated identified microbes will be serially diluted and a particular load of microbial concentration will be standardized along with the exposure time to the breast cancer cell line
All the experiments will be performed through the Sulforhodamine B SRB assay which is a colorimetric method used to determine cell viability or cytotoxicity
In this assay cells will be fixed with 10 percent Trichloroacetic acid TCA for 1 hr followed by staining with 004 percent SRB and washed with 1 percent acetic acid
At the end the optical density will be measured by suspending the stained cells in Tris base at 510nm
Additionally with the help of different in vitro assays the effect of the tumor microbiome on the TNBC cell line can be determined
For instance

1 Clonogenic assay to check the proliferation and colony formation ability of cancer cells
MDA MB 231 cells will be counted through a hemocytometer and almost 1000 cells will be seeded in every well of a 6 well plate
The next day a standardized concentration of microbes will be added to the wells
After 7 days the colonies formed by the cells will be fixed with 4 percent Glutaraldehyde and fixation will be performed with crystal violet
After washing with dH2O and drying the colonies will be counted and plotted in figures

2 Scratch assay to monitor the cellular migration
MDA MB 231 cells will be counted through a hemocytometer and almost 2 x 10 to the power 5 cells will be seeded in every well of a 12 well plate
After achieving 95 percent confluency a standardized concentration of microbes will be added to the wells
A vertical and horizontal scratch will be made through the 200uL tip the migration of cells will be taken at 6 12 and 24 hours time intervals

3 Live cell imaging analysis to check the morphological changes of cancer cells i e size and shape
The morphology of cells at different microbial concentrations will be measured through EVOS live cell imaging software and the area perimeter circularity index and aspect ratio will be calculated through ImageJ software
All the aforementioned assays will be analyzed through the comparative study of TNBC cell lines in the presence or absence of microbes

Furthermore cancer cells with the highest growth rate will be used to conduct further immunofluorescence IF studies
For the comparative study of different aspects of cellular changes in the presence and absence of microbes various IF staining methods will be used e g DAPI Nucleus Phalloidin Actin filament Annexin V PI Apoptosis

Detailed protocol of immunofluorescence staining
1 Seed the cells in a 9 mm coverslip
Next day treat the cells with optimum microbial concentration after 12 hrs of treatment wash the cells with 1X PBS twice and fix the cells with 4 percent Paraformaldehyde for 30 min
2 Discard the 4 percent Paraformaldehyde and again wash with PBS twice permeabilize with Triton X 100 for 30 min
Wash the cells through 1X PBS twice and blocking will be performed through 1 percent BSA for 45 min
3 Wash the cells through 1X PBS twice and add 2 uL of Actin staining in PBS for 40 min
4 Again wash the cells through 1X PBS twice and add 1 uL of DAPI staining for 5 min
5 Dry the coverslip add mounting media and start visualizing and taking images

Eventually the role of the microbiome on TNBC cancer progression will be validated through additional in vitro experiments based on the experimental results.

Definition of endpoints and their correlation with PFS

Objective 1:
A biomarker that indicates a specific drug target may be used to select patients for a clinical trial in which Progression-Free Survival (PFS) is the primary endpoint. If the biomarker is present, it suggests that the treatment is more likely to be effective, potentially resulting in a longer PFS. Therefore, the identification of differentially expressed proteins (biomarkers) from lymph node metastatic TNBC samples will help in patient stratification and improving therapeutic targeting and clinical outcomes.

Objective 2:
This objective will be conducted entirely through in vitro experiments using TNBC cell lines. Microbes will be isolated from human tissue samples, and the impact of tumor-associated microbes on the progression of breast cancer cells will be studied through various functional assays in cell culture. Since no human or clinical interventions are involved in this objective, no clinical endpoint is applicable or required.