Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Summary

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

Abstract

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Introduction

Developing efficacious cancer therapeutics has proven to be extremely challenging. Cancer cell lines and tumor explants - as well as xenografts have been used in cancer research for over half a century^1,2,3. To date, the molecular analysis of drug sensitivity and resistance in both established cancer cell lines and patient-derived xenografts (PDX) is indispensable. However, testing of compounds in established cancer cell lines is not often predictive of in vivo efficacy, and corresponding in vivo studies in animals, especially in PDX models, is very expensive and time consuming. The limitations of these model systems, namely the inability ....

Protocol

NOTE: Patient tissue procurement was authorized through institutionally review board (IRB)-approved Biospecimen and Clinical Protocols (Protocol numbers 09-121 and 11-041, respectively) at Memorial Sloan Kettering Cancer Center.

1. Tissue Procurement

1. Procurement of patient primary tumor/metastasis
   NOTE: To date, this protocol has been performed on surgically removed pancreatic, gastric and breast tumor types, as well as, lymphoma metastases.
   1. Direct the Surgical team to deliver the specimen by courier or pneumatic tube system to the Pathology department in tightly sealed and sterile leak-proof plastic spec....

Results

For this study, the ex vivo technique was used in a correlative analysis of therapeutic sensitivity/resistance of a heat shock protein 90 inhibitor (Hsp90i). In a preclinical assessment of this Hsp90i, the breast cancer primary tumor, an ER+ invasive ductal carcinoma (IDC), and associated lymph node metastasis were analyzed ex vivo for treatment response (Figure 1). Multiple 200 μm serial sections were treated with vehicle only and increasing doses (0.25, 0.50, 1.0 and .......

Discussion

Cancer biologists face significant challenges when attempting to develop efficacious therapeutic strategies. Testing drugs in development on established cancer cell lines cannot accurately reflect in vivo response and in vivo experiments on PDX models are labor intensive and very expensive. Given the above, the application of ex vivo techniques of primary patient tumors^{14, 15} is now positioned beside the molecular analysis of established cancer cell lines and patient-derived xenograf.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Vibratome Leica VT1000s	Leica	14047235613
UltraPure agarose	Invitrogen	16500500	Prepare 4% and 6% before use
Injector blade	Ted Pella	121-4
MEM with Penicillin  + Streptomycin	Media Core Facilities (MSKCC)		The media is prepared at Memorial Sloan Kettering Cancer Center
Scalpel no. 10	Thermo Scientific	31-200-32
Disposable forceps	Cole-Parmer	84011182
Embedding mold	Electron Microscopy Science	70181
FBS (heat inactivated)	Gemini	100106
24 well plates	Corning	3524
Formalin (10%)	Sigma Diagnostics	SDHT501128
16% Formaldehyde solution	Thermo Scientific	28908
Embedding microsettes	Simport	M503-2
Ethanol (70%)	Fisher Scientific	A405P-4
Waterbath	Fisher Scientific	15-462-2SQ
Microwave	General Electric	ModelJES2051DNBB
Adhesive (Ethyl Cyanoacrylate)	Sigma-Aldrich	E1505-5G
10 mm dishes	BD Falcon	353003
15 ml tubes	BD Falcon	352096