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 to inform on the influence of the native microenvironment in tumor progression and response to therapeutic strategies, has led the research field to develop additional methods to compliment these analyses. Of recent, heightened attention is being paid towards ex vivo analysis of patient tumor explants ^{4, 5} due to the greater understanding that cancer therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment ^{6, 7} a feature that cannot be recapitulated by traditional culturing methods and/or PDX. Ex vivo analysis in the above context (i.e., influence of adjacent surrounding tumor cell microenvironment) implies assessment of viable primary tumor/metastasis sections, rather than ex vivo analysis of cellular isolates ^{8, 9}.

We report here on an ex vivo technique (i.e., precision-sliced fresh tissue sections) of both patient primary tumors and associated metastases (i.e., lymph nodes) that faithfully informs on response (IC50), off-target effects and allows for molecular analysis of resistance and feedback mechanisms. Additionally, a correlative analysis of therapeutic sensitivity/resistance versus biomarker and gene expression profile can be performed in an effort to identify patients more likely to respond to the experimental drug of interest (i.e., high drug response matches patient with particular biological profile). Application of the ex vivo technique and assessment in a multi-parameter fashion is movement towards patient selection and overall improvement of clinical outcomes.

Ex vivo treatment response analysis could become a standard tool in the preclinical and clinical development of cancer therapeutics and is envisioned as a step towards a personalized medicine approach in therapeutic development strategies.

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 specimen bag within sterile spill-proof container. Direct the Surgical team to transport the specimen in the fresh state with no formalin or fixative.
   2. Direct the pathologist or pathologist assistant to harvest the fresh specimen using sterile technique, which includes use of sterile gloves and instruments under a laminar flow hood.
   3. Record the harvesting time of the procured specimen, which is kept rigorously under 30 min from the completion of the surgical procedure. Taking into consideration effects of cold ischemia, do not take samples from resected specimens with a cold ischemic time of > 30 min¹⁸.
   4. Direct the Pathology Department personnel to remove a primary tumor specimen of approximately 0.5 cm³ to 1.0 cm³ in a laminar flow hood to maintain a sterile environment. When possible, choose tumor tissue from the periphery of the index lesion to avoid potential frank central necrosis (cell death).
      NOTE: The necrotic tissue may be grossly recognizable by any of the following criteria: loss of color or paleness of the tissue; loss of strength in which necrotic tissue is soft and friable; a distinct demarcation between the necrotic and viable tissue.
   5. Direct the pathologist or pathologist assistant to provide peripheral tissue that is in excess (surgical discard) after immediate sampling of the tumor for diagnostic evaluation. Place the specimen in a 15 ml sterile conical centrifuge tube containing approximately 5 ml of minimum essential media (MEM) containing 1% antibiotics (penicillin and streptomycin).
   6. If available (e.g., lymph node of axillary tail of mastectomy specimen), procure a grossly positive associated lymph node specimen of the same size (0.5 cm³ to 1.0 cm³) and compare to drug response of primary tumor. Like the primary tumor specimen, place the associated lymph node specimen in a 15 ml sterile conical centrifuge tube containing approximately 5 ml of minimum essential media (MEM) containing 1% antibiotics (penicillin and streptomycin).
   7. If size of surgical specimen permits (e.g., mastectomy specimen), remove (distant from primary tumor) a sample of normal tissue (i.e., normal dense/fibrous breast parenchyma) and place in a 15 ml sterile conical centrifuge tube containing approximately 5 ml of minimum essential media (MEM) containing 1% antibiotics for transfer purposes only. Following transfer to the laboratory facilities, transfer the ‘normal’ specimen to a cryovial, ‘snap’ freeze and store in a -80 °C freezer for future molecular analyses.
   8. Place all specimens on wet ice and immediately transport to the laboratory space for ex vivo fresh tissue sectioning. Do not section the normal specimen but rather store in a -80 °C freezer for future molecular analyses. Transfer a portion of the primary tumor and associated metastasis to a cryovial, ‘snap’ freeze and store in a -80 °C freezer for future molecular analyses.
2. Procurement of tissue from clinical studies (e.g., optional core needle biopsies (CNB))
   NOTE: We obtain tumor tissues from patients enrolled on clinical protocols who give their consent to undergo a CNB of an accessible tumor metastasis prior to the start of therapy. CNBs, to date, have been performed on patients with breast, marginal zone lymphoma, mantle cell lymphoma, metastatic paraganglioma, cervix (squamous cell) and ovarian cancers.
   1. Have clinical personnel from Interventional Radiology or relevant department perform a pre-treatment core needle biopsy and place the specimen in a 15 ml sterile conical centrifuge tube containing approximately 5 ml of minimum essential media (MEM) containing 1% antibiotics (penicillin and streptomycin).
   2. Place the biopsy specimen on wet ice and immediately transport to the laboratory space for ex vivo fresh tissue sectioning.

2. Preparation of Materials for Sectioning and Vibratome Settings

1. Preparation of Materials for Sectioning
   1. Make a 4% agarose solution by adding 4 g of agarose to 100 ml of PBS. Autoclave (cycle settings: liquid, 121 °C, 30 min, 15 pounds per square inch) the solution before first use. For subsequent uses, place the 4% agarose in a microwave to liquefy (High heat for 30 sec intervals) prior to each use and cool appropriately to ~ 25 °C (i.e., warm to touch) mindful that agarose solidifies at RT.
   2. Use the 4% agarose as a non-permanent tissue embedding media. When the 4% agarose has cooled sufficiently and is still in a liquid state, with sterile forceps place the tissue specimen in an embedding mold and pour in the 4% agarose.
   3. Position the tissue specimen close (~ 2 mm distance) but not directly on the cutting surface wall of the embedding mold. The 4% agarose will begin to solidify quickly, therefore appropriately position the tissue specimen quickly. During sectioning, keep the 4% agarose in a 55 °C water bath to keep in solution.
   4. To a 24-well plate for section analysis, add 1 ml of complete media (i.e., MEM + 10% fetal bovine serum + 1% antibiotics) to each well. Keep the 24-well plate at RT (~25 °C) throughout the duration of sectioning.
2. Preparation of Vibratome Settings
   1. Using the vibrating microtome, set the speed at which the blade approaches the specimen [0 (0.00 mm/sec) to 10 (2.5 mm/sec)] and the frequency of the blade [0 (0 Hz) to 10 (100 Hz)]. Adjust the settings according to the texture of the tissue (i.e., density/firmness).
   2. For a primary breast tumor of invasive ductal carcinoma, adjust settings to 6 and 3, speed and frequency respectively. For less dense/firm tumor tissue, decrease speed and increase frequency accordingly.
   3. Set the section thickness within the instrument limits (1 – 999 µm). For these analyses, set the section thickness at 200 µm.
   4. Mount the agarose-embedded specimen using a thin layer of liquid adhesive on the specimen disc that is submersed in a reservoir of media that is encased in wet ice.

3. Tissue Sectioning, Treatment & Analyses

1. Remove the precision-sliced sections immediately upon sectioning using sterile forceps and place in a multi-well plate with 1 ml of complete media. When sufficient sections are acquired for response and correlative analyses, place the multi-well plate for treatment response in a tissue culture incubator set at standard tissue culturing conditions.
2. Take multiple control sections that bookend the treated sections in an effort to confirm dose-dependent response as opposed to cultured artifacts. Perform tissue sectioning rapidly (~30 – 40 min), well within any detrimental effects of cold ischemic time (~2 – 4 hr)¹⁸. Perform treatment of sections only following a 1 hr acclimation period.
3. Following the 1 hr acclimation period, add increasing doses (therapeutically relevant; e.g., 0 – 10 μM concentrations) of drug to the sections and house under standard tissue culture conditions for the treatment period. For this study, use 24 and/or 48 hr assessment periods. However, control (vehicle only) tissue sections will remain viable, as tested in this study, for periods up to and including 96 hr assessment periods.
4. Following treatment, remove all sections under sterile conditions and formalin-fix by placing the tissue section in a tube containing either 10% buffered formalin and place at RT O/N or in a tube containing 4% paraformaldehyde (dilute 16% paraformaldehyde from a vial with PBS for a final concentration of 4%) and place at RT for 2 hr.
   1. Fix larger tissue sections (e.g., ~6 mm x 4 mm) 200 μm thick in 10% buffered formalin O/N at RT. Fix smaller sections (e.g., 1 mm x 1 mm) 200 μm thick in 4% paraformaldehyde for 2 hr at RT.
      NOTE: For efficient penetration of fixative the volume of fixative should be 20x the volume of tissue. Given that this protocol results in extremely small tissue sizes (e.g., 6 mm³ tissue = 6 μl volume), for assurance of proper fixation use 1 ml of fixative for all tissue sizes.
   2. Following fixation have the tissue specimens paraffin embedded by the Pathology Department personnel. Section specimen paraffin blocks onto charged slides to be hematoxylin and eosin stained and assessed immunohistochemically (e.g., apoptosis).

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